Fluorescent polybranched probes for detecting bacteria and/or fungi in vitro and in vivo

ABSTRACT

A probe is provided comprising a core and a plurality of probe elements; each probe element within the plurality of probe elements extending from the core and comprising a fluorophore and a binding moiety. Methods of use of the probe and kits comprising the probe are also provided.

FIELD OF THE INVENTION

The invention relates to the field of molecular probes, morespecifically to molecular probes for the detection of micro-organismssuch as bacteria and fungi.

BACKGROUND OF THE INVENTION

There is a growing burden of bacterial infections worldwide and accuratediagnosis remains a cornerstone to providing accurate treatment.

Patients within hospitals and in healthcare generally, are at risk ofbacterial infections. Hospital acquired infections (HAI) are becomingmore common, and the ability to respond to such infections rapidly andaccurately, is increasingly important. Where the patients have an activeand healthy immune system, the dangers posed by HAI are reduced.However, the immune system of patients that are seriously ill are oftenat least partially compromised, making the patient especially vulnerableto HAIs.

Ventilated patients in critical care, and indeed all immunocompromisedpatients, are especially vulnerable to HAI, and one of the mostdevastating HAI remains ventilator associated pneumonia (VAP). VAPremains notoriously difficult to accurately diagnose and inappropriatetreatment has been shown to be harmful to patients. Accordingly, VAP hasa high mortality rate, significant morbidity and remains a burden onhealthcare resources. In terms of diagnosis, the gold standard remainspulmonary biopsy, which is an invasive and rarely utilised investigationowing to the intrinsic invasive nature of the test. Other methods suchas bronchoalveolar lavage remain controversial, while non-culturemethods have not had any significant impact or robust validation.Clinical signs of fever, increased oxygen dependence and tachycardiaremain as non-specific means of detecting inflammation or acute lunginjury and therefore, alternative approaches are required that willallow the accurate and timely diagnosis of VAP, which will allowimmediate healthcare decisions to be made and, with appropriate therapy,improve patient outcomes.

Currently clinicians are faced with significant uncertainty in relationto when and if to commence antibiotic treatment, the choice of agents touse, and if treatment begins, when to de-escalate therapy. These issuesare barriers to effective antibiotic stewardship because of the provenassociation between delayed and inadequate antibiotic therapy andadverse clinical outcomes.

Molecular imaging technologies can allow the use of bacterial specifictracers and when combined with imaging modalities such as positronemission tomography (PET), have the ability to delineate infective fromsterile sites. This approach, however is not be applicable for somepatient groups, such as intensive care cohorts where a point of carediagnostic test would be required, and the administration of radioactiveagents can be problematic and restrictive. These also involve radiationand are not readily applicable to imaging outside hospital settings.

An alternative approach in the art is the application of optical probesto allow direct visualisation of a target area of a patient, through theuse of an endoscope. WO 2003/079015 in the name of Visen Medical, Inc.,discloses optical imaging probes for identifying and characterisingnormal and diseased tissues with regards to altered metabolic activity.

WO 2012/136958 in the name of the present applicant discloses brancheddyes to allow visualisation of cells in vivo by an increase influorescence when the dye is internalised by specific cell types.

However, there remains a need for improved optical probes and imagingmethods that will allow in situ, point of care determination of whethera patient's condition is due to an infection, and if so, what is thecausative agent of the infection.

Furthermore, there remains a need for improved optical probes that areretained at the target cell membrane, for example, during challengingconditions, such as in the presence of surfactants.

Therefore, an object of the present invention is to provide improvedprobes and imaging methods suitable for rapid and accurate point of carediagnosis, and to provide optical probes that are better retained bytheir target.

STATEMENTS OF THE INVENTION

According to a first aspect of the invention, there is provided a probecomprising a core and a plurality of probe elements; each probe elementwithin the plurality of probe elements extending from the core andcomprising a fluorophore and a binding moiety.

During use, the probes of the invention are typically delivered to atarget area and bind to any bacteria within the target area. Preferably,the binding moiety of each of the plurality of probe elements isavailable to bind to bacteria, and therefore, if bacteria is present inthe target area, the probe is bound to the bacteria by each, or at leastthe majority, of the binding moieties of the plurality of probeelements.

Optical probes known in the art are typically retained poorly in atarget area in vivo where challenging conditions can lead to degradationor oxidation of the probes, and/or protease breakdown, and as a result,the probes are unable to remain bound to their target to allow reliabledetection of the target. For example, optical probes used for imagingwithin the lung where there are high surfactant concentrations,typically do not allow their targets to be detected. Further challengeswithin the lung include “washing off” of probes by the circulating fluidtherein.

Surprisingly, the inventors have found that the probes of the inventionare more stable to oxidation, degradation and protease activity thanprobes known in the art, and that such probes allow reliable detectionof bacteria in vivo, even in challenging conditions such as those foundin the lung.

By the term “core” we refer to a common moiety that joins the pluralityof probe elements to form a single unit. Accordingly, the core could bea single atom, or comprise a functional group, a saturated orunsaturated hydrocarbon chain or a polyglygol (linear, branched, orcyclical), a peptide sequence, or a polymer.

Preferably, the plurality of probe elements comprises at least two probeelements, at least three probe elements, at least four probe elements orat least five probe elements. For example, the plurality of probeelements may comprise three probe elements.

The binding moiety may selectively bind to at least some bacteria, andnot bind to animal cells, such as mammalian cells, for example, or doesso weakly. Accordingly, the binding moiety may be a bacteria bindingmoiety. The bacteria binding moiety may bind to substantially allbacteria but not to animal cells, or does so weakly. The bacteriabinding moiety may selectively bind to substantially gram-negativebacteria, but not bind to gram-positive bacteria, or animal cells suchas mammalian cells, or does so weakly.

The binding moiety may selectively bind to fungi, and not bind to animalcells, such as mammalian cells, for example. Accordingly, the bindingmoiety may be a fungi binding moiety and may bind to fungi hyphae.

The binding moiety may selectively bind to at least some bacteria and toat least some fungi, and not bind to animal cells such as mammaliancells. Accordingly, the probe may allow the detection of fungi and/orbacteria in a target area. For example, the binding moiety may beadapted to bind to fungal hyphae of A. fumigatus.

The binding moiety of one or more of the probe elements may be aubiquicidin moiety, such as the full length ubiquicidin (SEQ ID NO.1) ora fragment or variant thereof. The binding moiety may be a fragment ofubiquicidin comprising at least 10 consecutive amino acids, or at least12 consecutive amino acids of ubiquicidin. For example, the bindingmoiety may be the ubiquicidin fragment of amino acids 29 to 41(UBI₂₉₋₄₁, SEQ ID NO.2). The binding moiety may be a ubiquicidin moietycomprising one or more substitutions. The or each substitution may be aconservative substitution, and have little or preferably, no effect onthe bacteria binding properties of the ubiquicidin. The or eachsubstitution may provide stability to the ubiquicidin moiety againstdegration or oxidation. For example, the binding moiety may be UBI₂₉₋₄₁comprising a substitution of a norleucine amino acid for the originalmethionine amino acid (UBI_(29-41NIe), SEQ ID NO.3).

Ubiquicidin is a mammalian anti-microbial peptide present in airwayepithelial cells, gut mucosa and in macrophages. Ubiquicidin bindsspecifically to the cell membrane of prokaryotes such as bacteria anddoes not bind to mammalian cells.

The binding moiety of one or more of the probe elements may selectivelybind to gram positive bacteria and may not substantially bind togram-negative bacteria. Preferably, the binding moiety of one or more ofthe probe elements selectively binds to gram-negative bacteria and doesnot substantially bind to gram-positive bacteria. Accordingly, inembodiments where the binding moiety of one or more of the probeelements selectively binds to gram-negative bacteria, the probe of theinvention may be randomly distributed in a target area that does notcomprise gram-negative bacteria, and be localised to the cell membranesof any gram-negative bacteria present in the target area. Therefore, theprobe of the invention may be adapted to selectively indicate thepresence, or absence, of gram-negative bacteria in a target area.

For example, the binding moiety of one or more of the probe elements maybe a polymyxin moiety, such as full length polymyxin (SEQ ID NO.4), or afragment or variant thereof. Polymyxin binds selectively to gramnegative bacteria, and therefore, a bacterial binding moiety comprisinga polymyxin moiety will selectively bind the respective probe element togram negative bacteria only, and thereby allow detection of any gramnegative bacteria within the target area. The binding moiety may be afragment of polymyxin comprising at least 6 consecutive amino acids, orat least 8 consecutive amino acids of polymyxin. The binding moiety maybe a polymyxin moiety comprising one or more substitutions. The or eachsubstitution may be a conservative substitution, and have little orpreferably, no effect on the gram-negative bacteria binding propertiesof the polymyxin moiety. The or each substitution may provide increasedstability to the polymyxin moiety against degradation or oxidation.

The binding moiety may be adapted to bind to at least some fungi. Thebinding moiety may be adapted to bind to fungal hyphae. For example, thebinding moiety may be adapted to bind to fungal hyphae of A. fumigatus.

Accordingly, the probe of the invention may be used to label fungi. Forexample, the probe of the invention may be delivered to a target areaand the probe may bind to any bacteria or fungi hyphae in the targetarea. Upon illumination of the target area with an appropriatewavelength of light to excite the or each fluorophore of the pluralityof bacteria binding probes, any bacteria and/or fungi within the targetarea will be labelled. The bacteria may be differentiated from thefungal hyphae visually, for example.

Therefore, the probe of the invention, may be used to determinebacterial or fungal infections, for example.

Preferably, each probe element within the plurality of probe elementscomprises the same fluorophore.

However, alternative embodiments of the invention may comprise probeelements having different fluorophores. For example, a probe of theinvention may comprise three probe elements, and the first probe elementmay comprise a first fluorophore, the second probe element may comprisea second fluorophore and the third probe element may comprise a thirdfluorophore.

Preferably, the fluorophore of each probe element within the pluralityof probe elements is an environmentally sensitive fluorophore, such thatthe intensity or quantum yield of fluorescence of the fluorophoredepends on the surroundings of the fluorophore. For example, the quantumyield or intensity of the fluorophore may be different in a free aqueousenvironment than when the fluorophore is in a hydrophobic environment,such as within a cell membrane. Preferably, the quantum yield orintensity of the fluorophore is higher in a hydrophobic environment,such as within a cell membrane. Accordingly, the intensity of lightemitted by the fluorophore increases when the fluorophore is at leastpartially within a cell membrane, such as when the probe element isbound within a cell membrane of bacteria, for example. Therefore, thefluorophore may be used to fluorescently label cells via their cellmembranes.

The fluorophore of one or more of the probe elements within theplurality of probe elements may be 7-nitrobenz-2-oxa-1,3-diazole (NBD),malachite green, a styryl-based dye, Cascade Yellow, prodan (aka1-Propanone, 1-(6-(dimethylamino)-2-naphthalenyl), Dansyl (aka.5-(dimethylamino)naphthalene-1-sulfonyl), Dapoxyl, PyMPO (aka.1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-Methoxyphenyl)Oxazol-2-yl)Pyridinium,pyrene and diethylaminocumarin, or derivatives or variants thereof.

Preferably, the fluorophore of at least one probe element within theplurality of probe elements is a NBD moiety. More preferably, thefluorophore of each probe element within the plurality of probe elementsis a NBD moiety. NBD has been found by the inventors to be aparticularly suitable fluorophore for the probe of the invention,providing strong fluorescence when the probe is bound to bacteria, and alow background fluorescence, allowing clear and reliable labelling ofbacteria compared to other fluorophores tested.

The fluorophore of one or more of the probe elements within theplurality of probe elements may have a long fluorescent lifetime and thelong fluorescent lifetime of the fluorophore allows the detection of theprobe over background autofluorescence. For example, during use, thefluorescence of the fluorophore may have a lifetime significantly longerthan the autofluorescence of the background of a target area such thatthe fluorescence from the fluorophore is readily distinguishable fromthe fluorescence of the background.

For example, the fluorophore is azadioxatriangulene (ADOTA) dye ordiazaoxatriangulene (DAOTA), or derivatives thereof.

Suitably, one or more fluorophores of adjacent probe elements within theplurality of probe elements may quench the fluorescence of one or moreof the other fluorophores of the probe. Therefore, the fluorophores ofthe probe may self-quench.

Preferably, in embodiments where the one or more flurophore of adjacentprobe elements within the plurality of probe elements self-quench, theself-quenching is prevented or reduced when the environment of the probeis changed. For example, the inventors have surprisingly found that inembodiments where the fluorophore of each probe element of the pluralityof probe elements is NBD, the probe emits low or no significantfluorescence in an aqueous environment due to the NBD moietiesself-quenching, but surprisingly, when the probe is in a hydrophobicenvironment, such as within a cell membrane, the NBD moieties are nolonger able to self-quench and the probe fluorescence is high.Accordingly, the probe of the invention may selectively fluoresce onlywhen it is bound to bacteria, and therefore, provides a relatively lowbackground fluorescence. Advantageously, this selective self-quenchingof the fluorophores within the probe improves the signal to noise ratiowithin the target area and provides clearer and more reliable detectionof bacteria. Furthermore, due to the higher concentration offluorophores on the probe (a plurality of fluorophores compared to asingle fluorophore), the fluorescent signal produced by probe when boundis higher, further improving the signal to noise ratio.

The binding moiety may be at the distal end of the probe elementfurthest from the core.

The binding moiety may be at the proximal end of the probe element andadjacent to the core, and the fluorophore of the probe element mayconnect to the core via the binding moiety. Alternatively, the bindingmoiety and the fluorophore of a probe element may be connected to thecore by a common connector and each of the binding moiety and thefluorophore may extend away from the connector.

The fluorophore may be connected to the binding moiety directly.Alternatively, the fluorophore may be connected to the binding moiety bya spacer. The spacer may be a saturated or unsaturated hydrocarbonchain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, apoly ether or similar. The spacer may be a peptide. In embodiments wherethe spacer is a peptide, the peptide may be 1-10 amino acids in length,1-20 amino acids in length, or 1-30 amino acids in length.

The probe may comprise a quencher connected to the core by a cleavablelinker; the fluorophore of one or more of the probe elements within theplurality of probe elements may be substantially fluorescently quenchedby the quencher when the core is connected to the quencher by thecleavable linker; wherein the core, and thereby the plurality of probeelements, is separated from the quencher when the cleavable linker iscleaved.

In embodiments where the fluorophores of the probe do not self-quench,the provision of a quencher that may be separated from the fluorophoresby cleavage of the linker allows the fluorophores to be quenched unlessa cleaving agent is present, which cleaves the linker. Accordingly, theprobe may allow detection of the presence of the cleaving agent bysignificant fluorescence of the fluorophores of the probe.

Preferably, the linker is dimensioned such that the quencher issufficiently close to the fluorophore of at least one of the pluralityof probe elements to quench the fluorophore. Typically, the quencher isless than 10 nm away from the fluorophore of at least one of the probeelements. Preferably, the quencher is less than 5 nm away from thefluorophore of at least one of the probe elements.

Preferably, the cleavable linker comprises an enzyme cleavable peptidesequence, and the linker is cleaved when a cleaving enzyme cleaves theenzyme cleavable peptide sequence.

Accordingly, cleavage of the enzyme cleavable peptide sequence typicallycorresponds to cleavage of the linker, and thereby cleavage of thequencher from the probe element. Accordingly, in embodiments where thecleavable linker comprises an enzyme cleavable peptide sequence, theterm “cleavage of the linker” refers to cleavage of the enzyme cleavablepeptide sequence unless stated otherwise.

The cleaving enzyme may be produced or expressed by the indigenous cellswithin the target area. The cleaving enzyme may be produced or expressedby additional cells produced by the patient that have migrated to thetarget area, such as leukocytes, for example neutrophils. The cleavingenzyme may be produced or expressed by an infective agent in the targetarea, such as a bacterial or fungal cell, for example.

Preferably, the cleaving enzyme is elastase and cleavage of the enzymecleavable peptide sequence is indicative of the presence of elastase.Typically, the elastase is neutrophil elastase and the elastase isproduced or expressed by neutrophils. Typically, the elastase is theactive form of the enzyme capable of proteolytic cleavage. Neutrophilstypically target sites within the body that are undergoing aninflammatory response, either pathophysiologically or part of normalfunction. Therefore, the presence of neutrophils in a target area withina patient is indicative that the tissue or a portion of the tissuewithin the target area is inflamed.

Therefore, an increase in fluorescence, or fluorescence of thefluorophore of at least one of the probe elements resulting from theseparation of the quencher and fluorophore by cleavage of the linker, isindicative of the presence of neutrophil elastase. Neutrophil elastaseis produced by neutrophils, and therefore the presence of neutrophilelastase is indicative of the presence of neutrophils and therefore,inflammation of the tissue within the target area. Accordingly, theprobe of the invention may be adapted to indicate inflammation of tissuewithin a target area.

Human elastase, such as human neutrophil elastase (HNE), and otherelastases or other enzymes such as proteases, typically have one or moretarget sequences to which they bind and cleave. Accordingly, the enzymecleavable peptide sequence will typically comprise the or a targetpeptide sequences of the cleaving enzyme to be detected, and the personskilled in the art would be able to select an appropriate peptidesequence for a given cleaving enzyme.

Typically, the enzyme cleavable peptide linker is a peptide sequencethat is a or the cleavage site of the cleaving enzyme. Preferably, theor each cleavage site of the cleaving enzyme comprises multiple aminoacids. In embodiments where the cleaving enzyme is elastase, preferably,the enzyme cleavable peptide sequence comprises the amino acid sequenceAAPV (i.e. alanine-alanine-proline-valine, or Ala-Ala-Pro-Val) orEEINIeRR. The enzyme cleavable peptide sequence may comprise one or moreadditional amino acids either side of the sequence AAPV in positions xand/or y, such as xAAPVy, xAAPV, or AAPVy, for example.

Alternatively, the cleaving enzyme may be a matrix metalloproteinase(MMP), such as MMP-9, and the enzyme cleavable peptide sequence maycomprise G-P-K-G-L-K-G. The cleaving enzyme may be proteinase 3 and theenzyme cleavable peptide sequence may comprise V-A-D-C-A-D-Y. Thecleaving enzyme may be cathepsin G and the enzyme cleavable peptidesequence may comprise A-A-P-F, or F-V-T-Gnf-S-W (whereGnf=4-guanidine-L-phenylalanine). The cleaving enzyme may be a caspaseand the enzyme cleavable peptide sequence may comprise D-E-V-D.

In alternative embodiments, the cleavable linker may be cleaved by areactive oxygen species, such as superoxide (O₂ ⁻) or hydrogen peroxide(H₂O₂), generated by the presence of bacteria, or inflammation processessuch as activated neutrophils. Accordingly, a reactive oxygen speciesmay be a cleaving agent. For example, the linker may be a modifiedboronic acid based linker, such as that described in J. Am. Chem. Soc,2014, 874, Roger Y. Tsien.

Optical probes known in the art typically are either fluorescent at alltimes (so-called “always on” fluorophores), and it is the location ofthese probes that is determined, or the probes change their fluorescencewhen their environment is changed, be that the removal or separation ofa quencher (for fluorescence resonance energy transfer, or FRET, basedprobes, for example), or being internalised by a cell.

However, in embodiments where the plurality of fluorophores of theplurality of probe elements do not self-quench, and the probe comprisesa cleavable linker, the probe of the invention advantageously changesits fluorescence due to whether the cleavable linker has been cleaved bythe cleaving agent, and the location of the probes when observed canindicate the presence of bacteria. Accordingly, the probes of theinvention are able to provide specific and more detailed informationthan optical probes known in the art. For example, the probes areadapted to indicate whether the cleaving agent is present (if the probefluoresces, or the colour of fluorescence changes the cleavable linkerhas been cleaved by the cleaving agent) and, if the cleaving agent ispresent, whether bacteria and/or fungi is present, indicated by thelocalisation of the probe to the bacteria and/or fungi hyphae and anincrease in fluorescence of the probe, for example.

The quencher may be a dark quencher. A dark quencher is a moiety that isable to accept energy from an excited fluorophore and dissipate thatenergy non-radiatively, typically as heat or acoustic energy, forexample. Therefore, when the fluorophore/quencher pair are sufficientlyclose together and are irradiated with a wavelength of light that iswithin the excitation spectra of the fluorophore, the quencherdissipates the energy absorbed from the light by the fluorophore in anon-radiative manner and no fluorescence is observed. In this way, thedark quencher suppresses the fluorescence of the fluorophore.

In embodiments where the quencher is a dark quencher, the quencher maybe methyl red, dimethylaminoazobenzenesulfonic acid (DABSYL), Iowa blackFQ or Iowa black RQ (Integrated DNA Technologies, Inc. Iowa, USA), BHQ1,BHQ2 or BHQ3, for example.

The quencher may be a fluorescent quencher. A fluorescent quencher is amoiety that is able to accept energy from an excited fluorophore and toradiate that energy. Accordingly, fluorescent quenchers are typicallyfluorophores, and emit light at a different wavelength to that emittedby the fluorophore from which they have accepted energy. In this way,the change in colour of fluorescence is indicative of the relativeproximity of the quencher to the fluorophore.

In embodiments where the quencher is a fluorescent quencher, thequencher may be rhodamine, tetramethylrhodamine or a derivative thereof,such as carboxytetramethylrhodamine (TAMRA) for example, fluorescein, ora derivative thereof, cyanine fluorophores or boron-dipyrromethene(BODIPY) fluorophores.

In embodiments where the quencher is a fluorescent quencher, thequencher may be adapted to label neutrophils. Accordingly, inembodiments where the cleavable linker comprises an enzyme cleavablepeptide sequence, the probe of the invention may be adapted to indicatethe presence of a cleaving enzyme, such as elastase, to label theneutrophils that are producing the enzyme, and to label any bacteriathat may be present.

For example, in embodiments where the cleaving enzyme is neutrophilelastase, the fluorescent quencher may be taken up by neutrophils thatare activated by the presence of bacteria or by stimuli such as calciumionophore, for example. Accordingly, the fluorescent quencher mayselectively label neutrophils within the target area. The inventors haveobserved that fluorescent quenchers, such as TAMRA, are selectivelytaken up by neutrophils and are not taken up, or are taken up to a muchlesser extent, by bacteria and by other inflammatory cells, such asmonocytes, and the inventors speculate that this observed difference inuptake of the fluorescent quencher may be due to the higher endocyticand pinocytic activity in highly phagocytic cells such as neutrophils.

The fluorophore of the probe element may form a FRET pair with thequencher. Typically, the fluorophore and the quencher are chosen as apair to ensure that they have appropriate excitation and emissionspectra for the transfer of energy from the fluorophore to the quencher(i.e. they form a FRET pair). For example, typical fluorophore/quencherpairs include Cy3/Cy5, Cy3/QSY21, fluorescein/tetramethylrhodamine,fluorescein/methyl red, cyan fluorescent protein (CFP)/yellowfluorescent protein (YFP), etc. Further examples of FRET pairs may bereadily identified by the skilled person.

In embodiments where the fluorophore of at least one of the probeelements within the plurality of probe elements is NBD, the quencher ispreferably a moiety that is able to accept energy from NBD to form aFRET pair with the NBD. Preferably, the quencher is methyl red ortetramethylrhodamine or a derivative thereof. For example, the quenchermay be carboxytetramethylrhodamine (TAMRA).

In embodiments comprising a cleavable linker, the binding moiety maybind to bacteria and/or fungi prior to cleavage of the cleavable linker.Alternatively, the binding moiety may bind to bacteria and/or fungiafter cleavage of the cleavable linker.

For example, the probe of the invention may be delivered to a targetarea and the probe may bind to any bacteria or fungal hyphae in thetarget area. Upon illumination of the target area with an appropriatewavelength of light to excite the fluorophore of the probe element, anybacteria and/or fungi within the target area will be labelled. Thebacteria may be differentiated from the fungal hyphae visually, forexample.

Typically, the probes of the invention are operable to be used to detectbacteria and/or inflammation in the target area. The target area may bea portion of tissue within a patient, and the method may be carried outin vivo. For example, the target area may be a portion of the lung of apatient, and the method may be carried out using a bronchoscope to bothdeliver the probe to the target area, to deliver light to the targetarea, and to detect fluorescence from the target area. Alternatively,individual instruments may be used to deliver the probe to the targetarea, to deliver light to the target area and to detect fluorescencefrom the target area.

For example, fluorescence may be detected from the tissue of a patientusing optical emission microscopy (OEM), such as fibered confocalfluorescence microscopy (FCFM).

The target area could be on the skin of a patient, in joints, in thecirculatory system, the digestive system or the reproductive system, andthe probes may be delivered and/or observed via an endoscope, forexample.

Alternatively, the target area may be a portion of a cell culture, atissue sample such as a biopsy sample, or a liquid sample such as abodily fluid sample, and the method may be carried out in vitro.

Preferably, the patient is a human patient. However, the patient may bea non-human animal such as equine, ovine, bovine, or rodents, forexample.

The probe may comprise a secondary label. For example, the core of theprobe may comprise a secondary label, or one or more of the plurality ofprobe elements may comprise a secondary label. The secondary label maybe a fluorophore. The fluorophore of the secondary label may bedifferent to the fluorophore of the or each probe element. The secondarylabel may be a radioactive label. The radioactive label may comprise aradionucleotide. The radioactive label may comprise one or more of ¹⁸F,⁶⁴Cu, ⁶⁸Ga, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ⁹⁰Y, ¹⁷⁷Lu, ¹¹C, ¹⁴C, ³H, ³²P, ³³P,¹⁸⁶Re, ¹⁸⁸Re, or ⁸⁶Zr. The secondary label may be a magnetic resonancelabel or core. The magnetic resonance label or core may be Fe, Mn, orGd, for example.

Accordingly, the probe may have dual- or multi-modality, and may be usedin optical imaging, radioactive imaging, or in magnetic resonanceimaging.

In further embodiments, the probe may comprise a targeting element thatis adapted to be internalised by a target species. The targeting elementmay comprise a fluorophore. The fluorophore of the targeting element maybe adapted to be internalised by the target species. For example, aprobe may comprise Cy5 and the probe, or a portion of the probecomprising the Cy5 once the linker has been cleaved, may be internalisedby any neutrophils in the target area to label them. Typically, theprobe comprises a quencher for the targeting element, and the targetingelement is separated from the quencher by the linker. In someembodiments, the probe may comprise a first portion and a second portionconnected by the linker. The first portion may comprise the probeelement and the quencher of the targeting element, and the secondportion may comprise the targeting element and the quencher for theprobe element. Accordingly, when the linker is cleaved, the probeelement and the targeting element are both separated from theirrespective quenchers.

For example, the probe element may comprise NBD (fluorophore) and QSY21,BHQ3 or BBQ650 (quenchers), and the targeting element may comprise Cy5,or sulfonated Cy5 (fluorophore) and methyl red or BHQ1 (quenchers).

Each probe element of the plurality of probe elements may extend fromthe core such that the probe is a branched, or multivalent probe, witheach probe element forming a separate branch of the probe. For example,the probe may have one of the following structures:

where

L=spacer group (e.g. C₃-C₁₀ alkyl, ((CH₂)₂O)_(x) where x=1-6)

F=fluorophore (e.g. NBD, fluorescein)

B=binding moiety (e.g. ubiquicidin moiety, polymyxin moiety)

C=core (e.g. carbon, —C(CH₂O(CH₂)₃)₃, NH₂CONHC(CH₂O(CH₂)₃)₃)

For example, in a preferred embodiment, the probe comprises three probeelements connected to a core, and has the general formulaNH₂CONHC(CH₂O(CH₂)₃-NBD-(CH₂)₅-UBI_(NIe))₃ where the core isNH₂CONHC(CH₂O(CH₂)₃)₃ and each probe element is:

NBD-(CH₂)₅CO-UBI_(NIe);

In an alternative embodiment, each of the three probe elements isNBD-(CH₂)₂O(CH₂)₂OCH₂-PMX;

The probe may have the structure:

A preferred embodiment of the probe has the structure:

According to a second aspect of the invention there is provided a methodof detecting bacteria and/or fungi in a target area, the methodcomprising the steps:

-   -   (1) providing a first probe according to the first aspect of the        invention;    -   (2) delivering the first probe to the target area;    -   (3) illuminating the target area with an appropriate wavelength        of light to excite the fluorophores of the first probe; and    -   (4) determining whether the first probe has labelled bacteria        and/or fungi within the target area.

Labelling of bacteria and/or fungi by the first probe within the targetarea is indicative of the presence of bacteria and/or fungi in thetarget area.

The target area may be tissue within a patient, and the method may becarried out in vivo. For example, the target area may be lung tissue ofa patient, and the method may be carried out using a bronchoscope toboth deliver the probe to the target area, to deliver light to thetarget area, and to detect fluorescence from the target area.Alternatively, individual instruments may be used to deliver the firstprobe to the target area, to deliver light to the target area and todetect fluorescent from the target area.

The target area may be a cell culture, a tissue sample such as a biopsysample, or a liquid sample such as a bodily fluid sample, and the methodmay be carried out in vitro.

Preferably, the patient is a human patient. However, the patient may bea non-human animal such as equine, ovine, bovine, or rodent, forexample.

In embodiments of the invention where the probe according to the firstaspect comprises a cleavable linker, the cleaving agent may be acleaving enzyme. The cleaving enzyme may be elastase, and significantfluorescence of the fluorophore of the probe element of the first probemay be indicative of the presence of the cleaving enzyme that cleavesthe linker. In embodiments where the patient is a human patient, thecleaving enzyme is preferably human neutrophil elastase (HNE). HNE istypically produced or expressed by neutrophils at the site of tissueinflammation. Accordingly, the presence of HNE is indicative ofinflammation of the surrounding tissue. Therefore, in embodiments wherethe cleaving enzyme is HNE, significant fluorescence of the fluorophoresof the plurality of probe elements of the first probe is indicative ofinflammation within the target area. For example, where the target areais within lung tissue of a patient, significant fluorescence of thefluorophores is indicative of inflammation of the lung tissue.

The method may allow detection of both sterile inflammation (i.e.inflammation that is not caused by infection) and infective inflammation(i.e. inflammation that is caused by a foreign body such as bacteria, orfungi, for example). Typically, significant fluorescence of the firstprobe without labelling of bacteria or fungi is indicative of sterileinflammation, and significant fluorescence of the probe with labellingof bacteria is indicative of bacterial inflammation. Significantfluorescence of the probe with labelling of fungi hyphae is indicativeof fungal inflammation.

By the term “significant fluorescence” we refer to the fluorescence ofthe fluorophore that may result from sufficient separation of thatfluorophore from the quencher of the first probe to prevent the quencherquenching the fluorescence of the fluorophore, that is above thebackground or, where present, autofluorescence in the target area. Theautofluorescence of the indigenous cells or tissue within the targetarea may have a shorter fluorescent lifetime than the fluorophore of thefirst probe. The autofluorescence of the indigenous cells or tissuewithin the target area may reduce over time at a faster rate than thatof the fluorophore of the probe. Accordingly, fluorescence observed inthe target area that reduces more slowly over time may be indicative ofthe first probe, and fluorescence observed in the target area thatreduces more quickly over time may be indicative of autofluorescence.

In many areas of healthcare, such as critical or intensive care, it isnot typically possible to use standard methods such as PET scans toidentify whether inflammation of the lungs, for example, is sterile orinfective, as the patients cannot be safely moved, especially when thosepatients are being ventilated. Accordingly, clinicians do not havesufficient information to confidently diagnose the inflammation andprescribe a suitable corrective course of action. For example, if theinflammation is sterile, giving the patient antibiotics will not help,and may have adverse side effects.

The method of the invention may be carried out by a clinician in situ atthe point of care, and determines whether bacteria and/or fungi ispresent in a target area, such as within the lungs, or a portion of thelungs of the patient. Accordingly, the method of the invention providesthe clinician with the information they need to confidently determinethe cause of any inflammation, and to determine the best course ofaction, such as giving an antibiotic to the patient, for example.

If bacteria is detected in the target area, and an appropriateantibiotic is given to the patient, the method may be carried out todetermine the efficacy of the antibiotic. For example, a reduction inthe number of bacteria, or the absence of bacteria in the target areatypically indicates that the course of treatment is effectively clearingthe bacterial infection. Similarly, if fungi is detected in the targetarea and an appropriate antifungal agent is given to the patient, themethod may be carried out to determine the efficacy of the antifungal.

The method may comprise the step of providing a second probe accordingto the first aspect comprising a binding moiety that specifically bindsto a subpopulation of bacteria. For example, the binding moiety of thesecond probe may bind to gram-negative bacteria specifically or may bindto gram-positive bacteria specifically.

The method may comprise the step of (5) delivering the second probe tothe target area. The method may further comprise the step of (6)illuminating the target area with an appropriate wavelength of light toexcite the or each fluorophore of the second probe. Step (5) and step(6) may be carried out after step (4), and the first probes may beallowed to dissipate or otherwise be removed from the target areabetween step (4) and step (5). In this way, when the target are isilluminated in step (6), substantially only the second probe will be inthe target area and therefore, any fluorescence observed may beattributed to the second probe. Therefore, the or each fluorophore ofthe second probe may the same or a different fluorophore to that of thefirst probe.

Alternatively, step (5) may be carried out between step (2) and step(3), and step (3) corresponds to step (6). Accordingly, both the firstprobe and the second probe are present in the target area when thetarget area is illuminated. Therefore, preferably a or each fluorophoreof the second probe is different to that of the first probe such thatthe fluorescence from the first probe and the fluorescence of the secondprobe are of different wavelengths, and can therefore, be differentiatedfrom one another. The provision of different fluorophores for each ofthe first probe and the second probe allows the presence of any bacteriaand/or fungi in the target area (shown by the localisation of the firstprobe to the cell walls of the bacteria or fungal hyphae), and whetherany bacteria are gram negative or gram positive (shown by whether thesecond probe has localised to the cell walls of the bacteria) to bedetermined. In embodiments where the probe comprises an enzyme cleavablepeptide linker connecting a quencher to the core of the probe, themethod of the invention allows the presence of inflammation to bedetermined (shown by the significant fluorescence of the first and/orsecond probes), the presence of any bacteria and/or fungi in the targetarea (shown by the localisation of the first probe to the bacteria orfungal hyphae), and the determination of whether the bacteria aregram-negative or gram-positive (shown by whether the second probe haslabelled the bacteria).

Therefore, the method of the present aspect of the invention mayadvantageously allow the cause of inflammation in a single test to beidentified, and in some embodiments, the determination of the presenceof sterile inflammation, thereby providing healthcare professionals withthe information they need in order to decide on the correct course ofaction for that patient.

The method may comprise the step of observing the target area underwhite or fluorescent light to determine the morphology of any infectiveagent (bacteria or fungi) identified in the target area. The method maycomprise the step of observing the target area under white orfluorescent light to identify microbes within the target area, and thefirst and second probes may be used to determine the identity of thosemicrobes.

Fluorescence from the first and/or second fluorophore may be imageddirectly by the fluorescence emitted from the first and/or secondfluorophore being directed onto a detecting device, such as acharge-coupled device (CCD) or a complementary metal-oxide-semiconductor(CMOS) device, for example. Fluorescence from the first and/or secondfluorophore may be imaged indirectly. For example, the fluorescence maybe converted into acoustic waves by using photoacoustic imaging.Photoacoustic imaging may allow high-resolution images of the targetarea to be generated. In embodiments where the first and/or secondfluorophore emit light in the near-infrared or infrared range ofwavelengths (˜700 nm-1 mm), the whole or substantially the whole, of thebody of the patient may be imaged. Alternatively, in embodiments wherethe the first and/or second fluorophore emit light in the visible rangeof wavelengths (˜390 nm-700 nm), a specific target area of the patientmay be imaged, and the light may be delivered to and received from thetarget area via a fibre optic, for example.

The method may comprise the step of observing the target area usingphotoaccoustic ultrasound to determine the identity of the infectiveagent (bacteria or fungi) detected in the target area. The method maycomprise the step of observing the target area using photoaccousticinstruments to identify microbes within the target area, and the firstand second probes may be used to determine the identity of thosemicrobes using direct fluorescent detection.

The first probe may comprise a ubiquicidin moiety, such as full lengthubiquicidin, or a fragment or variant thereof as the binding moiety.

The second probe may comprise a polymyxin moiety, such as full lengthpolymyxin or a fragment or variant thereof, as the binding moiety, andthe second probe may selectively bind to gram negative bacteria.

Accordingly, the method may allow the identification of sterileinflammation, bacterial and/or fungal inflammation, and gram negativebacterial infection (and by inference, gram positive bacterialinfection).

The invention extends in a third aspect to use of the probes of thefirst aspect to identify or label bacteria and/or fungi in a targetarea.

The probes may be used in vivo to identify or label bacteria and/orfungi in a target area. For example, the probes may be used in vivo inembodiments where the target area is a portion of tissue within apatient, such as lung tissue of a patient.

In embodiments where the probes are used in vivo, a bronchoscope orendoscope may be used to both deliver the probe to the target area, todeliver light to the target area, and to detect fluorescence of theprobes within the target area. Alternatively, individual instruments maybe used to deliver the first probe to the target area, to deliver lightto the target area and to detect fluorescent from the target area.

The probes may be used in vitro to identify or label bacteria and/orfungi in a target area. For example, the probes may be used in vitro inembodiments where the target area is a portion of a cell culture, atissue sample such as a biopsy sample, or a liquid sample such as abodily fluid sample.

In embodiments where the probes are used in vitro, fluorescence from theprobes may be detected using microscopy, such as confocal microscopy,for example.

A first probe of the first aspect may be used to identify or labelbacteria and/or fungi in a target area, and a second probe according tothe first aspect may be used to determine whether the identified orlabelled bacteria are gram-negative or gram-positive bacteria, whereinat least one binding moiety of the first probe binds specifically tobacteria and/or fungi, and at least one binding moiety of the secondprobe binds specifically to gram-negative or gram-positive bacteria. Forexample, at least one binding moiety of the first probe may be aubiquicidin moiety, and at least one binding moiety of the second probemay be a polymyxin moiety.

According to a fourth aspect of the invention, there is provided a kitof parts comprising one or more probes of the first aspect of theinvention and a suitable buffer within which the probe may be dispersed.

The kit of parts may comprise a first probe according to the firstaspect comprising at least one binding moiety that specifically binds tobacteria and/or fungi, and a second probe of the first aspect comprisingat least one binding moiety that specifically binds to gram-negative orgram-positive bacteria.

For example, the kit of parts may comprise a first probe comprising atleast one ubiquicidin moiety, and a second probe comprising at least onepolymyxin moiety.

Optional and preferred features of the first aspect are optional andpreferred features of the second, third and fourth aspects.

Embodiments of the present invention will now be described, by way ofnon-limiting example, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Fluorophore choice impacts on ability to image labelled bacteriawith Improved signal to noise for NBD constructs over Fluorescein (FAM).A) Bacteria (Methicillin sensitive staphylococcus aureus, MSSA),counterstained with FM-464 (shown in red), and incubated with FAM-UBI orNBD-UBI demonstrating an improved signal to noise ratio with NBD whichallowed the majority of bacteria to be detected (shown in green). Thiswas compared with FAM-UBI which detected a minor subset of clumpedbacteria. B) NBD-UBI also exhibits specificity for bacteria (Pseudomonasaeruginosa) over mammalian cells (white arrow).

FIG. 2: FAM-PMX does not allow sufficient signal to noise to detectbacteria in vitro. A) Pseudomonas aeruginosa (PA), counterstained withFM-464 (red) and incubated with A549 cells (human lung epitheial cellsin purple) demonstrate bacterial labeling is not seen above backgroundbut also there is no labeling of the epithelial calls. B) NBD-PMX(green) with a nuclear counterstain Syto 82 (red) allows bacteriallabelling and retains selectivity of labeling over mammalian cells(purple).

FIG. 3: Emission spectra of probes confirms increase in fluorescence inhydrophobic environments. A) Emission spectra shown for NBD-UBI_(dend)(5 μM) and NBD-UBI_(NIe) (15 μM) in the presence of PBS or DMSO(hydrophobic environment) when excited at 488 nm wavelength. B) Emissionspectra shown for NBD-PMX (10 μM) in the presence of PBS or DMSO(hydrophobic environment) when excited at 488 nm wavelength. Thefluorescence of both probes greatly increases under hydrophobicconditions as would be present upon binding to the target bacteria.

FIG. 4: NBD-UBI_(dend) labels bacteria selectively over mammalian cellsin vitro: A) Example images of a clinically relevant bacterial panelwith NBD-UBI_(dend) (5 μM; green) counterstained with the fluorescentgeneric cellular DNA dye Syto-82 (red) imaged by laser scanning confocalmicroscopy. B) Quantification of bacterial panel labellingNBD-UBI_(dend) 5 μM (optimal concentration) where every bacteria in thepanel is brighter when compared with MSSA and NBD-UBI_(NIe) 10 μM(optimal concentration) C) Flow cytometry data with unstained bacteria(red), NBD-UBI_(NIe) 15 μM (blue) and NBD-UBI_(dend) 5 μM labelledbacteria (orange). D) MSSA and NBD-UBI_(dend) (5 μM; green),counterstained with Syto-82 and merge showing human neutrophils (redarrow) and E) A549 cells (red arrow) demonstrating no labelling ofmammalian cells. Representative images shown, n≥3 for all experiments.

FIG. 5: NBD-PMX selectively labels Gram-negative bacteria in vitro.Example images of the bacterial panel with NBD-PMX imaged by laserscanning confocal microscopy. A) Bacterial panel with NBD-PMX 1 μM(green) and counterstain with syto-82 (red). Gram-positive bacteria(bounded by red box) display minimal/no labelling compared withGram-negative bacteria. B) Quantification of bacterial panel withNBD-PMX 1 μM with Gram-positive bacteria in white bars and Gram-negativein black bars, showing high intensity selective labelling ofGram-negative bacteria compared with Gram-positive bacteria. AllGram-negative bacteria showed a statistically significant increase overall Gram-positives. C) Flow cytometric evaluation of bacterial labellingwith NBD-PMX showing unstained bacteria (red) and NBD-PMX (blue)labelled bacteria. No significant labelling of Gram-positive bacteria(bounded by red box). D) MSSA and NBD-PMX 1 μM (green), counterstainedwith Syto-82 and merge showing human neutrophils (red arrow) and E) A549cells (red arrow) demonstrating no labelling of mammalian cells(reproduced from FIG. 2). Representative images shown, n≥3 for allexperiments.

FIG. 6: Ex vivo model of bacterial infection. A) Ovine lungs wereharvested, ventilated and placed in a neonatal incubator with ambienttemperature of 37° C. Pulmonary segments were instilled with PBS(control) or bacteria via bronchoscopy. Probes were then instilled andsegments imaged with fibered confocal fluorescence microscopy (FCFM)using 488 nm Laser Scanning Unit (LSU). B) Ex vivo ovine modeldemonstrates viable bacteria 5 hours following instillation. Bacteria(MSSA) instilled into segments of ovine lung and lavaged at 1, 3 and 5hours (n=3), plated for CFU/ml and counted following 16 hour incubation.

FIG. 7: NBD-UBI_(NIe) fails to label bacteria in the ovine lung. Therewas no consistent labelling of bacteria in the ovine lung confirmed bycounterstaining bacterial and imaging on a spectrally distinct imagingsystem. A: Lung segment with PKH660 labelled MSSA at 1 hourpost-instillation. B: Control segment (instilled with PBS and nobacteria) imaged at 660 nm. C) The same segment imaged at 488 nmfollowing 10 μM NBD-UBI_(NIe) instillation. However, the same experimentwith counterstained PKH660 labelled K. pneumoniae (Gram negative) shownin panel D in purple, with NBD-PMX added demonstrating the same punctatesignal.

FIG. 8: Representative FCFM images of bacteria in the distal lungdemonstrating a distinctive punctate pattern. A) Instillation of PKHlabelled S. aureus, Calcein labelled S. aureus or GFP-expressing strainof S. aureus, generate a punctate pattern of fluorescence when segmentsare imaged using FCFM. Images used to generate positive control forsubsequent in situ labelling experiments. Representative images shown,n=3 for all experiments. B) PKH dyes do not stain by-stander cells.Confocal images showing epithelial cells in co-culture with bacteria (S.aureus) that have been counterstained with Syto-82 (red) as well asPKH660 (purple). Bystander mammalian cells (red arrows) are labelledwith Syto-82 that has leached from the labelled bacteria but there is notransfer of PKH660.

FIG. 9. NBD-UBI_(dend) detects bacteria in situ in the distal ovinelung. Representative FCFM images of NBD-UBI_(dend) showing that nopunctate signal is seen in control segments whereas the distinctivepunctate signal described in FIG. 8 above is seen above backgroundfluorescence in segments instilled with bacteria. Furthermore, agarosebeads alone show no fluorescence (control beads) but when beads arecoated with bacteria a signal is seen when labelled in situ in the ovinelung. Representative images shown, n≥3 for all experiments.

FIG. 10: NBD-PMX selectively labels Gram-negative bacteria in situ inthe ovine lung. A) Control segments and Gram-positive segments show nopunctate signal, whereas Gram-negative instilled segments show signalidentical to positive control. B) Lavage counts from segmentsdemonstrating no significant difference in counts between segmentsconfirming the presence of bacteria. C) Agarose beads alone show nofluorescence but when beads are coated with bacteria a signal is seenwhen labelled in situ in the ovine lung. Representative images shown,n≥3 for all experiments.

FIG. 11: NBD-UBI_(dend) and NBD-PMX are resistant to ‘wash off’ allowingdetection of bacteria in the ovine lung. Bacteria pre-labelled witheither NBD-UBI_(dend) or NBD-PMX were readily visualised by FCFM wheninstilled into the ovine lung. However, when bacteria pre-labelled withlinear NBD-UBI_(NIe) are instilled, no punctate signal is seen.Labelling was confirmed by imaging bacterial suspensions (left handpanels) before instillation into the lung and imaging by FCFM (righthand panels). Note the lower signal-to-noise ratio for NBD-UBI_(NIe)labelled bacteria in suspension. Representative images shown, n=3 foreach experiment.

FIG. 12: NBD-UBI_(dend) retains fluorescence when wash performed. A) Thebacterial panel was imaged by laser scanning confocal microscopy in thecontinued presence of NBD-UBI_(dend) (black) and following PBS wash(white). Quantification demonstrates fluorescence retention above linearNBD-UBI with MSSA in the continued presence of probe (red-line). n≥3,with three random field-of-views assessed for each experiment. B)Bacterial panel imaged with NBD-PMX in the continued presence of probe(black) or following PBS wash (white) demonstrating higher fluorescenceretention of all Gram-negative bacteria above NBD-PMX withGram-positives in the continued presence of probe. n≥3, with threerandom field-of-views assessed for each experiment.

FIG. 13: NBD-UBI_(dend) is stable in Acute Lung Injury BroncholalveolarLavage Fluid (ALI BALF). Matrix-Assisted Laser Desorption/IonizationTime-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis demonstratedstability of NBD-UBI_(NIe) in the presence of saline (arrow indicatescorrect peak seen at mass of 1949) but degradation in the presence ofALI lavage (no peak seen at mass 1949 but arrows show predictabledegradation compounds) when co-incubated for 30 minutes. By contrastNBD-UBI_(dend) remains stable when assessed by Fourier Transform MassSpectrometry (FTMS) (data shown represents a theoretical plot and anexperimental plot demonstrating the peaks correspond indicating presenceof compound). NBD-PMX is stable in ALI BALF. MALDI-TOF analysisdemonstrated stability of NBD-PMX in the presence of saline or ALIlavage (arrows indicates correct peaks seen at mass of 1291-1293) whenco-incubated for 30 minutes.

FIG. 14: NBD-UBI_(dend) retains higher fluorescence on bacteria in ovinelavage than eqimolar equivalent of linear. A) MSSA imaged by laserscanning confocal microscopy in the presence or absence of ovine lavagewith NBD-UBI_(NIe) (15 μM) and NBD-UBI_(dend) (5 μM). Both NBD-UBIcompounds have reduced fluorescence in ovine lavage but NBD-UBI_(dend)retains higher fluorescence intensity. B) In contrast, PA3284 imaged inthe presence or absence of ovine lavage with NBD-PMX (1 μM) demonstratesno significant reduction of fluorescence intensity. Representativeimages shown, n=3.

FIG. 15: NBD-UBI_(dend) and NBD-PMX successfully image bacteria in asurfactant-rich environment. Ratio of bacterial fluorescence intensity:surfactant fluorescent intensity quantified from confocal microscopyimages. For equimolar NBD concentrations NBD-UBI_(dend) and NBD-PMX havesignificantly higher bacterial: surfactant intensity than NBD-UBI_(NIe).

FIG. 16A: Elastase dependent bacterial labelling scheme. The fluorophoreon the bacterial binding component is quenched by a dark quenchermeaning that no fluorescence is seen upon binding to bacteria. Cleavageof the elastase specific sequence AAPV liberates the bacterial bindingcomponent (PV-NBD-UBI) from the dark quencher methyl red (MR).

FIG. 16B: Elastase dependant bacterial labelling in vitro. MSSA,counterstained with FM-464 (red), co-incubated with isolated humanneutrophils demonstrating bacterial labelling in the presence ofneutrophils (green), which is inhibited in the presence of an elastaseinhibitor.

FIG. 17A: Elastase dependent bacterial labelling scheme. The fluorophoreon the bacterial binding component is quenched by a differentfluorophore of a longer wavelength meaning that no fluorescence is seenupon binding to bacteria. Cleavage of the elastase specific sequenceAAPV liberates the bacterial binding component (PV-NBD-UBI) from thequencher (TAMRA). TAMRA reports the presence of activated neutrophils byendocytic uptake.

FIG. 17B: Elastase dependent bacterial labelling in vitro. Pseudomonasaeruiginosa (PA) counterstained with PKH 660 (purple) was incubated withTAMRA-AAPV-NBD-PMX and demonstrated no labelling in the presence ofunactivated neutrophils (top panel), bacterial labelling (shown ingreen) in an elastase dependent manner and labelling of activatedneutrophils (shown in red) in an elastase independent manner.

FIG. 17C: Elastase dependent fluorescence increase of TAMRA followingcleavage. Spectrophotometer data to demonstrate the unexpectedobservation of an increase in fluorescence of TAMRA-AA (cleavedcompound) compared to TAMRA-AAPV-NBD-PMX in an elastase dependentmanner, and inhibited with an elastase inhibitor (ex 525 nm, Em 580 nm).

FIG. 18: Image Processing Algorithms delineate ‘positive’ and ‘negative’frames from FCFM data (A) raw data for a frame that is deemed positiveor negative. (B) Without pre-processing, spot-detection is ineffective.Following estimation of background noise (C), high-pass filtering (D)and appropriate thresholding (E), the current algorithm detect minimalpunctate signal in negative frames (F).

FIG. 19: Automated Image Processing of entire FCFM videos (up to 3500frames) demonstrates detection of bacteria. A) Analysis ofNBD-UBI_(dend) videos with thresholding at 80 spots per frame and anarbitrary cut-off of 10% enables objective detection of bacteria using acombination of Laplacian of Gaussian (LoG) and Difference of Gaussians(DoG) positive frames. B) Analysis of NBD-PMX videos with thresholdingat 80 spots per frame and an arbitrary cut-off of 10% enables objectivedetection of) Gram status using a combination of LoG and DoG positiveframes. n=4 for all analyses, except S pneumoniae where n=3. Statisticalanalysis when compared to control.

FIG. 20: Bacteria detected in the presence of human lung tissueautofluorescence. A) FCFM images of human lung tissue with PA3284demonstrating elastin autofluorescence and no bacterial signal (left)and human lung tissue with PA3284 co-incubated with NBD-PMX (right)demonstrating bacterial signal. B) Live confocal microscopy images(merge) showing merge images of nucleic acid counterstain (red) with noNBD-PMX labelling. Bacteria in right panel can be clearly seen, labelledwith NBD-PMX and above the background fluorescence. C) The fluorescentlifetime of NBD is significantly longer than that of theautofluorescence of the indigenous cells of the lung, measured by laserexcitation at 488 nm and time resolved single photon countingspectrometer, and this can be used to identify the probes in vivo andincrease signal-to-noise of sensing and imaging.

FIG. 21: NBD-UBId_(end) labels bacteria and variably labels A. fumigatusbut not C. albicans imaged by confocal microscopy. Representative imagesof NBD-UBI_(dend) 5 μM demonstrating A: labeling of bacteria (Paeruginosa) and fungal hyphae of A. fumigatus (blue arrow) but not C.albicans (red arrow). Panel B demonstrates the variability of labelingon fungal hyphae.

FIG. 22: NBD-PMX labels bacteria and variably labels A. fumigatus butnot C. albicans imaged by confocal microscopy. Representative images ofNBD-PMX 5 μM demonstrating A: labeling of bacteria (P aeruginosa) andfungal hyphae of A. fumigatus (blue arrow) but not C. albicans (redarrow). Panel B demonstrates the variability of labeling on fungalhyphae. Data shown for 5 uM but similar results seen for 1 uM.

FIG. 23. Suspensions of C. albicans do not demonstrate increasedfluorescence when imaged with FCFM. Figure shows positive control of C.albicans labeled with Syto green and imaged in suspension but nolabeling without the probe, with NBD-UBI_(dend) or NBD-PMX with FCFM.

FIG. 24: A. fumigatus demonstrates fluorescence in a distinctive andcharacteristic manner when imaged with FCFM. Top panel is without theprobe demonstrating no intrinsic autofluorescence and bottom panels showdistinct pattern with NBD-UBI_(dend) and NBD-PMX.

FIG. 25: A. fumigatus demonstrates linear pattern of fluorescence in thepresence of ovine lung tissue, which is distinct from a bacterialsignal. Top panel shows the pattern of fluorescence with a bacterialsolution with the probe and bottom panels show A. fumigatus filamentswith NBD-UBI_(dend) and NBD-PMX on ovine lung.

FIG. 26: No haemolysis seen with NBD-UBI_(dend) or NBD-PMX up to 100 μM.Haemolysis assay (n=3) demonstrating no haemolysis on concentrations upto 100 μM. Positive control was 0.2% Triton-X and values corrected torepresent 100% haemolysis for Triton-X.

FIG. 27. A: NBD-PMX shows no organ toxicity in a 2 week singleinstillation model in mice. Representative histology images (×100) forNBD-PMX compared to PBS control animals at 48 hours and 14 days. Nodifferences from control were seen in any group (blindly scored by aconsultant histopathologist). B: NBD-UBI_(dend) shows no organ toxicityin a 2 week single instillation model in mice. Representative histologyimages (×100) for NBD-UBI_(dend) compared to PBS control animals at 48hours and 14 days. No differences from control were seen in any group(blindly scored by a consultant histopathologist).

FIG. 28. Fluorescence Life Time Imaging easily distinguishes lung tissuefrom bacteria. Left panel shows confocal at 488 nm excitation of lungand bacteria (dots) in lung tissue. FLIM (right panel) shows cleardifferences in imaging of lung tissue (green) and bacteria (blue).Imaging performed using NBD-UBI_(dend) and S. aureus on human lungtissue

SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

In the following description of example embodiments of the invention,binding moieties comprising a polymyxin moiety are given the code “PMX”,and binding moieties comprising a ubiquicidin moiety are given the code“UBI”. For example, embodiments of the invention that comprise aplurality of probe elements comprising the NBD and the modifiedubiquicidin fragment UBI_(NIe) (“Dendron Probes”) is referred to asNBD-UBI_(dend).

Choice of Probe Element Fluorophore

For the reporting of bacteria we synthesised a probe comprising theprobe element only, and substituting the methionine of the ubiquicidinfragment UBI₂₉₋₄₁ for a norleucine, “NBD-UBI_(NIe)”. This probe wascompared to the same bacterial detecting moiety with another ‘always on’fluorophore, fluorescein (FAM), “FAM-UBI_(NIe)”, and showed an improvedsignal-to-noise on live benchtop confocal microscopy for the NBDreported (FIG. 1). Furthermore, it was confirmed that the labelling isspecific to bacteria and not cell membranes in general. For example,FIG. 1 shows that isolated human neutrophils were not labelled by theNBD-UBI_(NIe) probe, whilst the bacteria were labelled by theNBD-UBI_(NIe) probe.

To confirm the same would be observed for the PMX bacterial detectingmoiety we constructed NBD-PMX and FAM-PMX demonstrating an improvedsignal-to-noise with NBD-PMX over FAM-PMX and confirm this construct isalso specific to mammalian cells (FIG. 2).

“Branched/Dendron” or “Multivalent” Probes

A probe comprising a core and three probe elements connected to the core(a “three branch” probe) was prepared (NBD-UBI_(dend)), each probeelement comprising a NBD-UBI_(NIe) moiety.

To confirm the fluorescent reporter NBD retains its characteristics whencoupled with our peptide moieties we measured the fluorescence of thecompounds in conditions to mimic a hydrophobic environment (DMSO).Linear NBD-UBI_(NIe), NBD-UBI_(dend) and NBD-PMX were excited at 488 nm(Biotek fluorescent plate reader) and demonstrated significant increasein fluorescence when the probes were in the presence of dimethylsulfoxide (DMSO) (hydrophobic environment) when compared to phosphatebuffered saline (PBS) (FIG. 3) confirming environmentally sensitivefluorescent reporting. NBD-UBI_(dend) demonstrated the same fluorescenceincrease as linear when using eqimolar concentrations of NBD.

A panel of bacteria which represent >70% of VAP causing pathogens(Chastre J et al. Am J Respir Crit Care Med. 2002 Apr. 1;165(7):867-903)(Gram-negative: P. aeruginosa (two strains), A. baumannii, S.maltophilia, K. pneumoniae, E. coli and H. influenzae. Gram-positive:Methicillin Resistant S. aureus (MRSA), Methicillin Sensitive S. aureus(MSSA) and S. pneumoniae) (strain list in Table 3 below) were labelledwith NBD-UBI_(dend). Labelling was observed with variable intensity(FIGS. 4A and B). Nevertheless all bacteria were brighter than MSSAlabelled with linear NBD-UBI_(NIe) (FIG. 4B) and on flow cytometryNBD-UBI_(dend) demonstrated increased labelling over linearNBD-UBI_(NIe) (FIG. 4C). Furthermore, NBD-UBI_(dend) did not label humanneutrophils or A549 cells supporting prokaryotic selectivity. (FIGS. 4Dand E).

NBD-PMX incubated with bacteria, demonstrated significantly higherfluorescence on Gram-negative bacteria (P. aeruginosa, A. baumannii, S.maltophilia, K. pneumoniae, E. coli and H. influenzae) thanGram-positive bacteria (MRSA, MSSA and S. pneumoniae) (p<0.05) onconfocal analysis (FIGS. 5A and B), which was confirmed by flowcytometry (FIG. 5C). Furthermore, there was no labelling of humanneutrophils or A549 cells. (FIGS. 5D and E).

NBD-UBI_(dend) and NBD-PMX were assessed for in situ specificity andsensitivity in an ex vivo ovine model of bacterial infection (FIG. 6).In this model, the instillation of PKH fluorescent dye-labelled bacteriainto the distal ovine lung and imaged with FCFM reveals a characteristicand distinctive pattern of punctate fluorescence in each field of view(FIG. 8). We observed identical patterns with Calcein-labelled bacteriaand Green Fluorescent Protein-expressing S. aureus which were instilledinto the lung and imaged with FCFM (FIG. 8). This pattern is reproducedidentically by instilling bacteria “pre-labelled” with NBD-UBI_(dend)and NBD-PMX into lung segments.

Following this thorough characterisation of the model and positivecontrols, we instilled PBS or VAP-relevant bacteria into distinctsegments within the ex vivo lung model, followed by the microdoseddelivery of probes according to the invention. We demonstrated that thelinear NBD-UBI_(NIe) could not label bacteria in situ despite theability in vitro (FIG. 7). For these experiments the assays wererepeated with counterstained gram-negative bacteria (K. pneumoniae)which demonstrated labelling with NBD-PMX (FIG. 7). However, In segmentsinstilled with bacteria we demonstrated the same signal as seen with the‘positive controls’ when NBD-UBI_(dend) is instilled (P. aeruginosa, S.aureus, E. coli and K. pneumonia) but minimal/no signal with PBScontrol, confirming the ability to label bacteria in situ and imageusing the FCFM system (FIG. 9).

In segments instilled with Gram-negative bacteria, P. aeruginosa(laboratory strain PA01 and clinical VAP isolate J3284), K. pneumoniaeand E. coli, we have demonstrated the same signal as in the ‘positivecontrols’ when NBD-PMX is instilled but no signal in segments with PBSor Gram-positive bacteria MSSA, MRSA and S. pneumoniae (FIG. 10). Inthese experiments we confirmed the equal density of Gram-positive andGram-negative bacteria in all of the segments imaged with NBD-UBI_(dend)and NBD-PMX by bronchoalveolar lavage (demonstrating no difference inCFU/ml between segments) and through counterstained bacteria imaged on aLSU at 660 nm.

To further demonstrate in situ bacterial detection and to assess theability of the probes to image bacterial aggregation, we embeddedbacteria in agarose beads which were then instilled into the lung.Microdosed probe instillation and FCFM imaging demonstrated thatbacterial beads are clearly and exclusively detected whereas controlbeads (beads without bacteria) are not (FIGS. 9 and 10).

In the distal lung, there is likely to be significant and rapiddissipation of the probes immediately after delivery. Therefore, it isimperative that probe-bacterial labelling remains persistent under theseconditions. NBD-UBI_(dend)-labelled bacteria retain labelling upon probe‘wash-off’, as is seen for NBD-PMX. When instilled into the ovine lung,bacteria pre-labelled with NBD-UBI_(NIe) are undetectable by FCFMwhereas bacteria pre-labelled with NBD-UBI_(dend) or NBD-PMX are readilyvisualised (FIG. 11). As such, this resistance to ‘wash-off’ representsa surrogate indicator of probe-bacterial affinity which appears to be anabsolute requirement for distal lung in situ labelling. Currently, bothNBD-UBI_(dend) and NBD-PMX are resistant to wash-off whilst most of theother structural variants do not. Hence, bacteria labelled with bothNBD-UBI_(dend) and NBD-PMX retain sufficient intensity of fluorescenceupon probe dissipation that occurs in the distal lung (FIG. 12).

Secondly we assessed stability of the probes in BALF from patients withALI by FTMS and MALDI-TOF MS analysis. NBD-UBI_(NIe), NBD-UBI_(dend) andNBD-PMX were incubated for 30 minutes with BALF (FIG. 13).NBD-UBI_(dend) and NBD-PMX demonstrate stability with peakscorresponding exactly to the predicted theoretical spectra of the intactprobes readily identified in both saline and BALF incubated samples.Predicted spectra of breakdown products were also not seen. By contrastbreakdown products of the NBD-UBI_(NIe) were seen indicating instabilityin lavage fluid. Thus, probe stability and retention of function in thecontext of the ‘inflamed’ highly enzymatically-active distal lungenvironment is a key determinant of in vivo utility. This was confirmedby imagining in the presence of ovine lavage, which demonstrated higherfluorescence intensity of NBD-UBI_(dend) over the linear compound, andno reduction in intensity for NBD-PMX when imaged in ovine lavage (FIG.14).

In vitro experiments were conducted using lung surfactant constituentsto investigate the ability of probes to preferentially detect bacteriain the presence of large amounts of surfactant. The nature of thefluorescent reporter (NBD) incorporated in NBD-UBI_(dend) and NBD-PMX,suggested the possibility of fluorescent activation in the hydrophobic‘rich’ surfactant environment. A suspension of surfactant constituentsin buffered saline was prepared (20 mg/ml, 65%dipalmitoylphosphatidylcholine, 30% phosphatidylglycerol, 5% palmiticacid with 1 mg/ml tyloxapol as a spreading agent) and incubated with andwithout A549 epithelial cell monolayer. Particles of surfactantconstituents seen to coat the epithelial cell-surface were fluorescentsuggesting the NBD fluorescence increases in this hydrophobic solution.We clearly demonstrate that NBD-UBI_(dend) and NBD-PMX both possessselectivity for bacterial labelling over lung surfactant constituents.At equivalent molarity, the NBD-UBI_(dend) has significantly improvedbacterial selectivity over linear NBD-UBI_(NIe) in the presence ofsurfactant constituents (FIG. 15). Despite a mechanism relying onfluorescence increase in hydrophobic environments by NBD, bothNBD-UBI_(dend) and NBD-PMX preferentially increase in fluorescence onbacteria over purified surfactant constituents, in keeping with theirability to image bacteria in the distal lung. Although the comparativedistribution and relative abundance of surfactant constituents in thelung is unknown it is entirely likely that this contributes to thebackground fluorescence that is observed upon probe instillation andthat retention of labelling in vitro in the presence of surfactant isrequired for the probe to image successfully, providing it issufficiently resistant to degradation and retains labelling uponwash-off.

Elastase (HNE) Sensitive Probes EXAMPLE 1

A first example of elastase sensitive probes (shown schematically inFIG. 16A) comprises methyl red (acting as a dark quencher) connected viathe peptide sequence AAPV (acting as the enzyme cleavable peptidesequence of the cleavable linker) to 7-nitrobenz-2-oxa-1,3-diazole (NBD)and the ubiquicidin fragment UBI₂₉₋₄₁ (together acting as the probeelement). Accordingly, the NBD fluorescence is quenched by the methylred and no fluorescent signal will be observed (i.e. the methyl red andNBD are acting as a FRET pair) and therefore, whether or not the probeis bound to any bacteria that may be present in the target area viaUBI₂₉₋₄₁, no signal is observed using confocal microscopy.

When the probe is in the presence of human neutrophil elastase (HNE),such as in inflamed tissue, HNE cleaves the peptide sequence of thelinker, to thereby free the probe element from the methyl red quencher.Accordingly, the NBD fluorophore is no longer quenched and produces afluorescent signal. In addition, due to the environmental sensitivity ofthe NBD, the signal produced is greatly amplified if the NBD is in ahydrophobic environment, such as within a cell membrane.

FIG. 16B shows confocal images for a sample comprising the probeincubated with a co-culture of bacteria and neutrophils. These imagesconfirm the ability of the probe to be cleaved by neutrophil derived HNEand to label bacteria. When the elastase inhibitor sivelestat was addedto the medium, no labelling was observed as the probe remains uncleaved(and therefore the NBD is quenched by the methyl red).

EXAMPLE 2

In a second example, the probe comprises carboxytetramethylrhodamine(TAMRA, acting as a fluorescent quencher) connected to NBD and polymyxin(acting as the probe element) via the peptide sequence AAPV (acting asthe enzyme cleavable peptide sequence of the cleavable linker).Accordingly, the NBD fluorescence is quenched by the TAMRA to give riseto a fluorescent signal from TAMRA (i.e. the TAMRA is accepting theenergy absorbed by the NBD and is itself fluorescing, TAMRA and NBD areacting as a FRET pair). Accordingly, whether or not the polymyxin hasbound to any bacteria that may be present, only a signal from the TAMRAis observed.

Once the probe has been cleaved by elastase, the bacteria are labelledby the probe element due to the fluorescence of NBD (FIG. 17A), whereasthe neutrophils, once activated, are labelled by the TAMRA moiety (FIG.17B). Furthermore, we demonstrate the fluorescence of the cleaved TAMRAcompound increases in an elastase dependent manner and are inhibitedwith an elastase inhibitor such as sivelestat (“S”, FIG. 17C).

Data Analysis

We have begun to develop bespoke image analysis/processing strategies toperform rapid real-time objective analysis of the large datasetsgenerated by the probe/FCFM platform. Unequivocal detection of bacteriaand the delineation of their Gram status will be achieved by employingthese image processing algorithms in real-time. A processing algorithmbased upon single frame analysis has been applied to entire videosequences (up to 3500 frames), and even at this early stage we are ableto unequivocally delineate bacterial presence and Gram status. Thesesignal and image processing algorithms will be rapidly iterated inreadiness for clinical application, and we expect significant componentsof machine-learning to be incorporated into further optimisation ofNBD-UBI_(dend) and NBD-PMX datasets.

FIG. 18 shows the stepwise processing for single positive and negativeframes that were initially chosen to develop the model, based on twoimage processing algorithms (The Laplacian of Gaussian (LoG) and theDifference of Gaussians (DoG)) commonly used to detect bright spots influorescence microscopy images in an objective computational manner.Spots are enhanced in an image by convolving with a LoG or DoG filter.Pre-processing is necessary to improve the accuracy by removing theinfluence of noise in the image. First, background noise is estimated bydividing an image into small windows (100 per frame) and thencalculating the standard deviation of the pixel intensities. High-passfiltering is used to decrease low frequency noise. Thresholding theresulting image at 3× standard deviation removes background noise andkeeps only the higher pixel intensity values. Finally, spots aredetected with LoG or DoG.

We have employed the method described above to analyse entire FCFM imagevideos (up to 3500 frames). With an initial thresholding limit of 80spots per frame to indicate a positive frame, we determined thepercentage positive frames per video and set an arbitrary ‘cut-off’ of20% as the threshold for a binary outcome of YES/NO. This showsunequivocal detection of bacteria and Gram status using NBD-UBI_(dend)and NBD-PMX respectively (FIG. 19).

To ensure the compounds can label bacteria above the normal human lungautofluorescence, bacteria were incubated with human lung tissue andimaged using FCFM and on benchtop confocal (FIG. 20) demonstrating thatbacteria can be detected, once labelled, above the level ofautofluorescence.

We have identified a number of determinants of distal lung in situlabelling with probes. These explain why the promising in vitro data forthe linear NBD-UBI_(NIe) probe did not translate to reproducible in situlabelling in the ovine lung. Structural variants were synthesised andassessed, exploring the structure-activity-relationship. The initialaims were to improve resistance to degradation and improvesignal-to-noise. Certain modifications which greatly improved a singlefunctional aspect such as stability (such as insertion of D-aminoacids/N-methyl or variants including exclusive D-amino acid variants) orlabelling intensity did not permit reproducible visualisation ofbacteria in the distal lung. We comprehensively assessed the UBIanalogues for stability in ALI BALF, in vitro labelling of bacteriausing live bench-top confocal imaging at 37° C. and in situ labelling inthe ovine lung.

A number of structural modifications have been undertaken which arebroadly divided into two groups:

(a) Increase the signal-to-noise ratio by examining differentenvironmentally-sensitive fluorophores or increasing the NBD-UBI_(NIe)payload.

We have assessed the utility of including two NBD fluorophores for eachUBI fragment and have also assessed this with an N-methylated amino acidvariant designed to enhance stability. We have assessed a series ofalternative environmentally-sensitive fluorophores, including malachitegreen and styryl-based dyes, with the aim of producing a highersignal-to-noise ratio, which may enable lower levels of bacterialdetection or detection at lower effective probe concentrations.

(b) Improve the resistance to proteolytic degradation.

We have assessed a number of compounds including variants incorporatingD-amino acids and N-methylated amino acids at selected positionsidentified by MALDI analysis of the parent NBD-UBI compound as sitessusceptible to proteolytic degradation. To reduce degradation withoutaltering the amino acid constituents we have synthesised variantsincluding PEG units at the amino and/or carboxy termini in order toblock degradation from the ends of the peptide sequence. We have alsosynthesised and assessed variants consisting entirely of D-amino acidsand D-amino acids with inversion of sequence with or without blockingPEG units. Furthermore, a cyclic variant of NBD-UBI_(NIe) has also beenassessed as well as further variants of this compound incorporatingN-methylated amino acids at selected positions identified by MALDIanalysis of the cyclic variant of NBD-UBI_(NIe) as remaining susceptibleto proteolytic cleavage

All compounds have undergone biological assessment. For stability wehave assessed each compound in the presence of 0.9% NaCl (Saline) orpooled lavage fluid from patients with acute lung injury and analysed bymatrix-assisted laser desorption/ionization (MALDI) or fourier transformmass spectrometry (FTMS). In vitro labelling was assessed on benchtopconfocal in the presence of compound with bacteria and labelling wascompared to NBD-UBI_(NIe) which served as a reference control forbacterial labelling. Where appropriate we have also assessed labellingof the compound on isolated human neutrophils and primary human celllines (A549 human lung adenocarcinoma cell line). For the ex vivo and invivo ovine lung experiments each compound was assessed in a control lungsegment (instilled with 2 ml PBS) or a bacterial segment (instilled with2 ml of 2 optical density of bacteria). Following bacterialinstillation, the compound was administered to the segment of interestand this was imaged by probe based FCFM.

Assessment of the different NBD-UBI_(NIe) variants has given us aclearer understanding of different mechanistic factors affecting thefunction of the probe in the lung environment, and specificallyclarified the reasons why, out of all the UBI variants, only theNBD-UBI_(dend) is able to image bacteria in the lung.

The alternative fluorophores were inferior to NBD for this application.A Malachite Green variant gave a much lower labelling intensity on thebacteria and although the Styryl-dye compounds exhibited an increasedintensity of labelling on bacteria these compounds had a decrease inselectivity over mammalian cells, most likely due to the propensity ofthe dyes themselves to enter lipophilic membranes overcoming thetargeting of the ubiquicidin moiety. Consequently, the styryl-dyevariants exhibited greatly increased off-target labelling in the ex vivolung and no bacterial signal was observed.

The D-amino acid variants as well as the variants with PEG blockinggroups at the ends of the peptide sequence exhibited reduced labellingin vitro and no labelling ex vivo. Despite improved stability, of thelinear UBI variants which retained function in vitro none of these wereable to image bacteria in the lung. We have obtained evidence that thewash-off properties of the probes, most likely related to affinityand/or the nature of subsequent insertion into the bacterial membrane,impact on whether or not they can be used to successfully image in thelung. The retention of labelling upon removal of probe solution wasinvestigated in vitro by confocal. With all of the linear UBI variantslabelling was lost completely upon wash-off. This suggests that, in thelung, labelling would be rapidly lost once the probe concentrationaround the bacteria decreased as a consequence of fluid dissipation.Bacteria were pre-labelled with these linear NBD-UBI_(NIe) compounds andsuccessful labelling was confirmed by imaging the bacterial suspensionpre-instillation. These suspensions were instilled into the ex vivo lungand when the segment was subsequently imaged by FCFM no bacterial signalwas detected (there is an inherent time-delay in change-over from theinstillation catheter to passage of the FCFM fibre into the samesegment). However when bacteria pre-labelled with NBD-PMX, which didn'tlose labelling upon wash-off when assessed by in vitro confocal, wereinstilled a bacterial signal was detected. The NBD-UBI_(dend) construct,as well as giving an increased bacterial signal at equimolarconcentrations, retained labelling upon wash-off. As predicted, whenbacteria pre-labelled with these compounds were instilled into the exvivo lung the bacteria were successfully imaged.

We tested the compounds ability to label non-bacterial pulmonarypathogens, including fungi. Within immunocompetent patients thedevelopment of VAP secondary to eukaryotic fungi is uncommon, andaccounts for <2% of VAP cases. The most commonly isolated pathogens areCandida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus).C. albicans colonises up to 50% patients in the ICU and colonisationincreases with antibiotic use. However, the development of invasivecandidiasis as a cause of VAP is rare and remains contentious. Indeedsome large clinical trials have excluded candida isolation in theirdiagnostic algorithms for VAP and post-mortem studies have demonstratedgrowth of candida from BAL is not a reliable marker of candida infectionin the immunocompetent patient. A fumigatus is also occasionallyisolated in 1-2% of respiratory samples in critically ill patients.However, of this 1-2% only 20% are believed to be pathogenic and theremaining 80% are believed to colonisation.

NBD-UBI_(dend) demonstrated no labelling of C. albicans, however, therewas variable labelling of A. fumigatus hyphae upon imaging (FIG. 21).Furthermore, imaging demonstrated the fungal hyphae are significantlylarger than bacteria and show a distinct morphological appearance.NBD-PMX demonstrated an identical pattern to NBD-UBI_(dend) when imagedon confocal with the retention of labelling of Gram-negative bacteria,variable labelling of A. fumigatus hyphae and no labelling of C.albicans (FIG. 22).

Finally, to detect if this pattern would be identified in lung tissue,A. fumigatus was co-cultured with ovine lung and the probes and imagedwith FCFM. This again demonstrated the size and pattern of the fungalhyphae to be distinct from a bacterial signal (FIG. 24). Similarexperiments were conducted with suspensions of A. fumigatus anddemonstrate a distinct characteristic, linear pattern of fluorescencewhich differs to a bacterial signal (FIG. 25).

NBD-PMX and NBD-UBI_(dend) were assessed for direct red cell toxicity bya haemolysis assay and demonstrated no red cell haemolysis up to 100 μM(FIG. 26). The compounds were assessed for toxicity in a rodent singledose, intratracheal toxicity assessment. Mice received a singleintratracheal administration of 100 μg/25 g mouse (3000 fold human doseassuming 100 μg delivered to a 75 kg man) of NBD-UBI_(dend) or NBD-PMX,or PBS control. Mice were then euthanized at 48 hours and 14 days (n=3per group).The data shows no toxicity for NBD-PMX (Table 2 and FIG. 27A)or NBD-UBI_(dend) (Table 1 and FIG. 27B).

TABLE 1 NBD-UBI_(dend) 48 hours 14 days PBS Control NBD-UBI_(dend) PBSControl NBD-UBI_(dend) Cytospin (% of 96.5/0.7/0 ± 96.4/3.6/0 ± ns100/0/0 ± 100/0/0 ± ns Mononuclear 0.7/0.67/0 2.0/2.0/0 0/0/0 0/0/0cells/ Neutrophils/Red Blood Cells) BALF (Cells/ul) 354.9 ± 66.6  477.1± 150.6 ns 361.1 ± 38.6  342.7 ± 6.5  ns PenH value  0.5 ± 0.04  0.5 ±0.06 ns 0.38 ± 0.03  0.5 ± 0.04 ns Creatinine (u/l) 8.0 ± 0.6 8.3 ± 0.7ns 12.7 ± 1.7  9.0 ± 1.5 ns Bilirubin (u/l) 2 ± 0 2 ± 0 n/a* 2 ± 0 2 ± 0n/a* ALT (u/l) 33.3 ± 3.9  24.0 ± 1.2  ns 39.3 ± 10.8 28.3 ± 1.9  ns ALP(u/l) 120.0 ± 6.2  222.3 ± 80.3  ns 178.3 ± 27.4  227.0 ± 27.1  nsAlbumin (u/l) 21.0 ± 1.0  22.3 ± 1.8  ns 24.3 ± 0.7  25.0 ± 1.2  ns ns =not significant. *Bilirubin for these time points was below 2 for allanimals.

TABLE 2 NBD-PMX 48 hours 14 days PBS Control NBD-PMX PBS Control NBD-PMXCytospin (% of 97.1/0.3/2.6 ± 96.1/0/3.9 ± ns 100/0/0 ± 93.0/0/2.3 ± nsMononuclear 2.9/0.3/2.6 3.9/0/3.9 0/0/0 2.3/0/2.3 cells/ Neutrophils/RedBlood Cells) BALF (Cells/ul) 243.1 ± 80.5  509.0 ± 270.2 ns 478.4 ±88.4  542.0 ± 84.7  ns PenH value 0.7 ± 0.1  0.5 ± 0.05 ns  0.4 ± 0.03 0.5 ± 0.07 ns Creatinine (u/l) 35.3 ± 28.9 16.3 ± 8.8  ns 8.7 ± 1.8 9.7± 0.3 ns Bilirubin (u/l) 2.4 ± 0.4 1.2 ± 0.4 ns 2.4 ± 0.3 1.2 ± 0.4 nsALT (u/l) 43.0 ± 14.0 36.0 ± 9.0  ns 32.3 ± 7.9  40.3 ± 4.8  ns ALP(u/l) 100.0 ± 18.7  107.7 ± 17.3  ns 89.0 ± 8.1  126.3 ± 8.5  ns Albumin(u/l) 24.7 ± 0.3  23.7 ± 0.3  ns 24.0 ± 0.6  25.3 ± 0.7  ns ns = notsignificant.

Methods of Synthesis of Probes Synthesis of Ubiquicidin Based ElastaseProbes (“Methyl Red (MR)-AAPV-NBD-UBI₂₉₋₄₁” and“TAMRA-AAPV-NBD-UBI₂₉₋₄₁”)

MR-AAPV-K(NBD)-PEG-OH (AL3-74) fragment was synthesised on solid-phaseemploying Fmoc-strategy, with standard amino acid coupling cycles (2×30min at rt) with DIC and oxyma in peptide grade DMF at ˜0.1 mM reagentconcentration. Fmoc deprotection steps were done in 20% piperidine inDMF (2×30 min). Between each step, the resin was washed with DMF, DCMand MeOH.

2 g of chlorotrityl polystyrene resin (loading ˜0.3 mmol/g) was treatedwith Fmoc-PEG-OH (3 eq) and DIPEA (6 eq) in anhyd. DCM (2 mL) for 3 h.After washing and Fmoc deprotection, the Fmoc-AAPVK(Dde) sequence wassynthesised as described above using, Fmoc-Lys(Dde)-OH, Fmoc-Val-OH,Fmoc-Pro-OH, and Fmoc-Ala-OH. After the sequence was completed, thesynthesis was continued with half of the resin (0.3 mmol scale) and Ddeprotecting group was orthogonally removed with NH₂OH/imidazole inNMP/DCM (2×90 min). The resin was treated with NDB-Cl (3 eq) and DIPEA(6 eq) in DMF (2×45 min). After Fmoc deprotection, the synthesis wascontinued in 0.15 mmol scale and Methyl Red was coupled to theN-terminus as described above. After washing, the fragment was cleavedoff the resin with TFA-TIS-H₂O (95:2.5:2.5) (30 min) and precipitatedwith cold ether to give AL3-74 (ESI−MS 1044.4 and 1066.4).

UBI₂₉₋₄₁ sequence was synthesised on Rink-amide ChemMatrix resin(loading 1 mmol/g) as described below. Next, AL3-74 (0.055 mmol) inanhyd. DMF (0.6 mL) was added to Ubi₂₉₋₄₁ on a ChemMatrix resin AL3-68(0.03 mmol), followed by addition of HBTU (0.055 mmol) and DIPEA (0.22mmol). The reaction mixture was shaken overnight covered from light.After filtration, the resin was washed with DMF, DCM and MeOH. The resinwas swollen with DCM and the probe was deprotected and cleaved off theresin with TFA/thioanisole/EDT/anisole (90:5:3:2) (3 h). The crudeprecipitated with cold ether and collected by centrifugation. Theproduct AL3-79 was purified by preparative HPLC with detection at 490 nmand gradient of H₂O-ACN with 0.1% formic acid as an eluent. MALDI-TOF MS2719.4, >95% HPLC purity.

TAMRA-AAPV-NBD-UBI₂₉₋₄₁ AL3-88 (Maldi-TOF MS 2881.5, >95% HPLC purity)was synthesised in similar manner expect fragmentTAMRA-AAPV-K(NBD)-PEG-OH (AL3-75) was coupled to the N-terminus ofUBI-based peptide on resin AL3-68.

Synthesis of Polymyxin-Based NeBac-Probe

TAMRA-AAPV-K(NBD)-PEG-OH fragment 3A was synthesised on solid-phaseemploying Fmoc-strategy, with standard amino acid coupling cycles (2×30min at rt) with DIC and oxyma in peptide grade DMF at ˜0.1 mM reagentconcentration. Fmoc deprotection steps were done in 20% piperidine inDMF (2×30 min). Between each step, the resin was washed with DMF, DCMand MeOH.

500 mg of chlorotrityl polystyrene resin (loading ˜0.3 mmol/g) wastreated with Fmoc-PEG-OH (3 eq) and DIPEA (6 eq) in anhyd. DCM (2 mL)for 3 h. After washing and Fmoc deprotection, theFmoc-AAPVK(Dde)-sequence was synthesised as described above using,Fmoc-Lys(Dde)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, and Fmoc-Ala-OH. After thesequence was completed, Dde protecting group was orthogonally removedwith NH₂OH/imidazole in NMP/DCM (2×90 min). The resin was treated withNDB-Cl (3 eq) and DIPEA (6 eq) in DMF (2×45 min). After Fmocdeprotection, 5(6)-carboxyTAMRA was coupled to the N-terminus asdescribed above. After washing, the fragment was cleaved off the resinwith TFA-TIS-H₂O (95:2.5:2.5) (30 min) and precipitated with ether. 3AESI−MS 1044.4 and 1066.4.

Next, to 3A (0.011 mmol) in anhyd. DMF (0.5 mL), HSPyU (0.011 mmol) andDIPEA (0.033 mmol) were added, and the reaction was stirred at rt for 1h. Boc-protected Polymyxin (15 mg, 0.012 mmol in 0.5 mL DMF) and DIPEA(0.033 mmol) were added, and the reaction mixture was stirred overnightcovered from light. DMF was evaporated, the crude dissolved into 1 mLTFA-DCM (1:1), and stirred for 90 min. TFA-DCM was evaporated, the crudeprecipitated with cold ether, and collected by centrifugation. Theproduct was purified by preparative HPLC with detection at 490 nm andgradient of H₂O-ACN with 0.1% formic acid as an eluent. Maldi-TOF MS2151.6 and 2173.6, 100% HPLC purity.

Synthesis of NBD-UBI_(dend) Synthesis of Monomer (5)

Synthesis required the preparation of the monomer (5) which wassynthesised in six steps¹ as shown in Scheme 1. Monomer (5) was preparedby the 1,4 addition of the hydroxy groups of1,1,1-tris(hydroxymethyl)amino-methane onto acrylonitrile, followed byamino group protection (Boc). Hydrogenolysis of the nitrile groups withPtO₂/H₂ gave (3) which was treated with DdeOH to give the tris-Ddeprotected amine (4). Following removal of the Boc protecting group, theisocyanate (5) was prepared following the procedure of Knölker.²

Fmoc-Rink Amide ChemMatrix Resin (6)

4-[(2,4-Dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Rinkamide linker) was attached to ChemMatrix resin (LV=1 mmol/g). Thus theFmoc-Rink-amide linker (0.2 mmol, 1 eq) was dissolved in DMF (4 mL) andethyl oximinocyanoacetate (Oxyma) (0.2 mmol, 1 eq) was added and themixture was stirred for 5 min. N,N′-Diisopropylcarbodiimide (DIC) (0.2mmol 1 eq) was then added and the resulting mixture was stirred for afurther 2 min. The solution was added to ChemMatrix resin (0.1 mmol, 1.0mmol/g, 1 eq) and shaken for 0.5 hour. The resulting resin was washedwith DMF (3×5 mL), DCM (3×5 mL) and MeOH (3×5 mL). The coupling reactionwas monitored by a quantitative ninhydrin test³.

The probe was synthesised on a ChemMatrix resin derivatized with anFmoc-Rink Amide type linker (Scheme 2). The linker (6) was loaded withmonomer (5) to give the tri-branched scaffold (7). Following the removalof the Dde groups (2% hydrazine in DMF) the appropriate Fmoc-Amino acidswere coupled sequentially followed by the attachment of4-PEG-7-nitrobenzofurazan N-hydroxysuccinimide ester (NBD-PEG-NHS) andcleaved from the resin using TFA/TIS/DCM (90/5/5).

General Procedure for the Fmoc Deprotection

To the resin (pre-swollen in DCM) was added 20% piperidine in DMF (5 mL)and the reaction mixture was shaken for 10 min. The solution was drainedand the resin was washed with DMF (3×10 mL), DCM (3×10 mL) and MeOH(3×10 mL). This procedure was repeated twice. The coupling reaction wasmonitored by a quantitative ninhydrin test³.

Isocyanate Coupling to Give (7)

To resin (0.30 mmol), pre-swollen in DCM (10 mL), was added a solutionof isocyanate (6) (920 g, 0.93 mmol), DIPEA (0.2 mL, 0.93 mmol) and DMAP(22 mg, 0.17 mmol) in a mixture of DCM/DMF (1:1, 5 mL) and the mixturewas shaken overnight and the reaction monitored by a quantitativeninhydrin test. The solution was drained and the resin was washed withDMF (3×20 mL), DCM (3×20 mL) and MeOH (3×20 mL) and ether (3×20 mL).(3×20 mL). The coupling reaction was monitored by a quantitativeninhydrin test³.

PEG Coupling—8-(9-Fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoicAcid (Fmoc-PEG-OH) Coupling

A solution of Fmoc-PEG-OH (3.0 mmol, 10 eq) in DMF (3 mL) and Oxyma (3.0mmol, 10 eq) was added and the mixture was stirred for 5 min. DIC (3.0mmol, 10 eq) was then added and the resulting mixture was stirred for afurther 2 min. The solution was added to pre-swollen resin (7) in DCMand the reaction mixture was shaken for 0.5 h. The solution was drainedand the resin was washed with DMF (3×10 mL), DCM (3×10 mL) and MeOH(3×10 mL). The coupling reaction was monitored by a quantitativeninhydrin test³.

Peptide Synthesis

Peptide Sequence: Thr-Gly-Arg-Ala-Lys-Arg-Arg-Nle-Gln-Tyr-Asn-Arg-Arg

A solution of the appropriate Fmoc-amino acid (3.0 mmol, 10 eq)(Fmoc-Arg(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-NIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH,Fmoc-Thr(tBu)-OH) and Oxyma (3.0 mmol, 10 eq) was added and the mixturewas stirred for 5 min. DIC (3.0 mmol, 10 eq) was then added and theresulting mixture was stirred for a further 2 min. The solution wasadded to pre-swollen resin in DCM and the reaction mixture was shakenfor 0.5 h. The solution was drained and the resin washed DMF (3×20 mL),DCM (3×20 mL) and MeOH (3×20 mL). The coupling reactions were monitoredby a quantitative ninhydrin test³.

7-Nitrobenzofurazan (NBD) Coupling

To a solution of NBD-PEG-NHS (3.0 mmol, 10 eq) in DMF (3 mL) was addedDIPEA (3.0 mmol, 10 eq). The resulting solution was added to resin (1eq), pre-swollen in DCM, and the reaction mixture was shaken for 0.5 h.The solution was drained and the resin washed with DMF (×3), DCM (×3)and MeOH (×3). The coupling reaction was monitored by a quantitativeninhydrin test³.

TFA Cleavage and Purification of Reporter NBD-UBI_(dend)

The resin (45 mg), pre-swollen in DCM, was treated with a cleavagecocktail of TFA/TIS/DCM (90/5/5, 300 μL) for 2.5 h. The solution wasdrained and the resin was washed with the cleavage cocktail and thesolution was removed in vacuo. The crude material was dissolved in aminimum amount of cleavage cocktail (50 μL) and added to ice-cold ether(7.5 mL). The precipitated solid (22 mg) was collected by centrifugationand the solvent removed by decantation and the precipitate was washedwith cold ether (3×5 mL). The precipitate was then purified bypreparative reverse phase HPLC and the required fractions were pooledand lyophilized to afford NBD-UBI_(dend).

Synthesis of NBD-PMX

The NBD-PMX probe was synthesised from its precursor Polymyxin B sulfatein four steps (Scheme 6). The probe and its intermediates weresynthesised using reported methods¹ with moderate modifications. Thefluorophore is incorporated as an amide coupling between the NHS esterof the NBD-PEG and the tetra-Boc polymyxin C. NBD-PMX probe is obtainedafter the TFA cleavage and HPLC purification.

Preparation of Compound B

Polymyxin B sulfate (10 g, 7.7 mmol, 1 eq) was dissolved in deionizedwater (200 mL) at a pH of 6.5 (use HCl aq solution to adjust the pH).Papain (1.5 g) was dissolved in water (25 mL) (same pH). The solutionswere combined and toluene (0.5 mL) was added, and the mixture was gentlystirred at 65° C. overnight. The mixture was then stirred in boilingwater for 5 min and the precipitate formed (denatured papain) wasremoved by centrifugation and filtration. The filtrate was concentratedin vacuo and freeze dried to give the crude product B in quantitativeyield. This step was carried forward to the next step without anyfurther purification. MS m/z 963.2 (100%, [M+H]⁺).

Preparation of Compound C

Crude B (5.5 g, 5.7 mmol, 1 eq) was dissolved in a mixture ofH₂O:Dioxane:Et₃N (150 mL, 1:1:1) and Boc-ON (4.52 g, 17.1 mmol, 3 eq)was added. The solution was stirred for 20 min at room temperature andthen quenched with methanolic ammonia (20 mL, 2M ammonia in MeOH). Thereaction was followed up by ELSD. Solvents were evaporated and theresulting mixture was subjected to silica gel chromatography column(MeOH:DCM, 15:85) to afford white solid B (1.7 g, 22%). MS m/z 1363.7(100%, [M+H]⁺).

N-(4-Nitrobenz-2-oxa-1,3-diazol-7-yl)amino-3,6-dioxaoctanoic Acid(NBD-PEG-OH):²

DIEA (850 μl, 5.00 mmol) and solid 8-Amino-3,6-dioxaoctanoic acid(NH₂-PEG-OH) (392 mg, 2.40 mmol, 1 eq) were added slowly, over an hour,to a solution of NBD-Cl (401 mg, 2.01 mmol) in methanol (20 mL) at 0° C.The reaction mixture was stirred overnight at room temperature. Thesolvent was evaporated and the remaining material was purified bychromatography on silica with DCM/MeOH (8:2) as the eluent to giveNBD-PEG-OH (400 mg, 1.23 mmol, 51%) as dark red oil. ¹H NMR (500 MHz,DMSO): δ 10.9 (s, 1H; COOH), 8.49 (d, J=8.5 Hz, 1H; CH NBD), 7.1 (s, 1H,NH), 6.23 (d, J=8.5 Hz, 1H; CH NBD), 4.25 (s, 2H), 3.93 (t, J=5.3 Hz,2H; CH₂), 3.80 (s, 4H), 3.72 (t, J=6.8 Hz, 2H; CH₂) ppm; MS (ESI−): m/zcalcd for C₁₂H₁₄N4O₇ [M−H]: 325.1; found: 325.2.

N-(4-Nitrobenz-2-oxa-1,3-diazol-7-yl)amino-3,6-dioxaoctanoic Acid,Succinimidyl Ester (NBD-PEG-NHS):

To a solution of NBD-PEG-OH (2.4 g, 7.4 mmol, 1 eq) in anhydrous DCM(500 mL) was added EDC.HCl (1.56 g, 8.18 mmol, 1.1 eq) and DIPEA (1.36mL, 10 mmol). After stirring the mixture for 10 min,N-hydroxysuccinimide (0.94 g, 8.18 mmol) was added and allowed to stirfor 16 h. The reaction mixture was diluted with DCM (250 mL) and treatedwith 5% aqueous citric acid (2×200 mL), sat. aqueous NaHCO₃ and brine.The organic layer was dried over Na₂SO₄, filtered and reduced in vacuoto afford product as dark brown solid (1.0 g, quantitative). The crudewas used for next step without further purification.

Preparation of F

A solution of NBD-PEG-NHS (466 mg, 1.1 mmol, 1 eq), DIPEA (384 μL, 2.2mmol, 2 eq) and amine C (1.5 g, 1.1 mmol, 1 eq) in DMF (150 mL) wasstirred at room temperature for 1 h and protected from light. Aftercompletion of the reaction (TLC), volatiles are removed under vacuum.The crude mixture was purified by flash chromatography (DCM:MeOH, 90:10)to afford dark orange/brown solid (1.2 g, 65%). HPLC (254 nm & 495 nm)Rt=7.80 min; m/z 1671.7 (25%, [M+H]⁺); 1693.9 (65%, [M+Na]⁺).

Preparation of NBD-PMX Probe

A solution of Boc-protected polymyxin F (150 mg, 0.09 mmol) in 20% TFAin DCM (2 mL) was vigorously stirred for 45 min at room temperature andprotected from light. The reaction mixture was evaporated in vacuo andthe resultant was dissolved in ether. Ether layer was decanted aftercentrifugation (3×2 mL). The resultant yellow/brown solid (40 mg,quantitative) was dried under vacuum. The crude product was purified bypreparative HPLC in MeOH/H₂O as gradient solvent system with 0.1% formicacid as an additive. The fractions collected from prep-HPLC were freezedried to afford red/orange solid (30 mg, 26% recovery from HPLC).

Characterisation: For analytical HPLC, a Poroshell 120 SB-C18, 2.7 μm,4.6×50 mm column was used with a diode array detector. For prep-HPLCmethod: Discovery C18 reverse-phase column (5 cm×4.6 mm, 5 μm) with aflow rate of 1 mL/min and eluting with H2O/MeOH/HCOOH (95/5/0.05) toH2O/MeOH/HCOOH (5/95/0.05), over 6 min, holding at 95% MeOH for 4 min,with detection at 254 and 495 nm and by ELSD. HPLC (495 nm): Rt=4.1 min;MS m/z 1271.7 (95%, [M+H]⁺); 1293.7 (100%, [M+Na]⁺); FTMS calc. 636.3282([M+2H]/2)⁺, found 636.3344.

Absorption/Emission: 467 nm/539 nm.

Solubility: Fully soluble in water.

Stability: stable at room temperature for >than 1 week.

Storage: Stored at −20° C. under inert atmosphere. Protect from light.

Biological Methods

Bacterial Growth: Bacteria used in assays include Pseudomonas aeruginosa(PA01-reference strain and J3284-clinical isolate from VAP patient),Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcusaureus (Inc. methicillin-resistant S. aureus (MRSA),methicillin-sensitive S. aureus (MSSA)), Klebsiella pneumoniae,Escherichia coli, Haemophilus influenzae and Streptococcus pneumoniae.

TABLE 3 Bacteria, strain reference and original source used inexperiments. Bacteria Strain Original Source Gram- P. aeruginosa ATCC47085 ATCC negative (PA01) bacteria P. aeruginosa J3284 ClinicalIsolate* A. baumannii J3433 Clinical Isolate* S. maltophilia J3270Clinical Isolate* K. pneumoniae ATCC BAA1706 ATCC E. coli ATCC 25922ATCC H. influenzae Clinical Isolate Clinical Isolate* Gram- MethicillinATCC25923 ATCC positive Resistant S. aureus bacteria (MRSA) MethicillinSensitive ATCC 252 ATCC S. aureus (MSSA) S. pneumoniae D39 NCTC 7466Health protection agency culture collection GFP fluorescent SRN6390-Gfp- Nottingham aureus EryR University (Gift from Professor PhilHill) *Gifts from Professor John Govan, University of Edinburgh.

All bacteria were grown on agar broth, chocolate agar or blood agarplates, stored at 4° C. For assays, a single colony of bacteria wastaken using an inoculating loop and added to 10 ml liquid broth in a 50ml Falcon Tube. This was transferred to an incubator at 37° C. for 16hours (for Streptococcus pneumoniae supplemented with 5% CO₂). Cultureswere either used as overnight cultures (stationary phase) or from thesecultures a sub-culture was taken (1:100) and the sample was grown untilthey entered mid log phase (reads of 0.5-0.6 optical density (OD) onspectrophotometer at 595 nm). The culture was then centrifuged at 4000rpm for 5 minutes and pellet resuspended in phosphate buffered saline(PBS). Following three washes this was reconstituted to 0.5 OD₅₉₅nm forconfocal assays, 0.1 OD₅₉₅nm for flow cytometry or 2 OD₅₉₅nm for ovineex vivo lung experiments (unless otherwise stated).

Bacterial Counting: Samples (prepared bacteria or lavage from ovine lungsegments) were vortexed briefly then serial dilutions (1:10) wereperformed to dilutions to the 8th dilution. The broth/blood agar platewas divided into quadrants with 5×20 ul drops in each quadrant. Thesewere incubated at 37° C. for 16 hours (for Streptococcus pneumoniaesupplemented with 5% CO₂) and plates were counted with data reported ascolony forming units per millilitre (CFU/ml).

Surfactant Constituent Synthesis: Surfactant 5 μg1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 2.5 μgL-α-Phosphatidyl-DL-glycerol sodium salt (from egg yolk lecithin; PG)were dissolved in 500 μl chloroform and evaporated under nitrogen to athin lipid film in a round bottom flask. The lipid film was rehydratedwith PBS at 48° C. for 1 hour with agitation (750 rpm) to generatemultilammelar vesicles (MLV). These were diluted 1:4 for use in confocalexperiments.

Agarose Bacterial Beads: Bacteria were grown to midlog phase in 400 mlTSB, pelleted by centrifugation and resuspended in 2 ml PBS. This wasmixed with 18 ml molten tryptic-soy agar (50° C.) and injected rapidlyinto vortexing mineral oil +0.01% Span 80, pre-warmed to 50° C. This wasthen rapidly cooled to 4° C. whilst continuing to vortex to allow thebeads to set. Bacterial agar beads were pelleted by centrifugation (20minutes, 3000 g) and washed in 0.5% sodium deoxycholate (SDC) in PBS (20minutes, 3000 g), followed by 0.25% SDC (20 minutes, 3000 g) in PBS,washed in PBS (10 minutes, 3000 g) and 3× PBS (5 minutes, 200 g). Beadswere resuspended at 50% v/v in PBS for instillation.

Neutrophil Extraction: Neutrophils were isolated from the peripheralblood of healthy human volunteers by dextran sedimentation followed bycentrifugation through discontinuous plasma-Percoll gradients.

A549 cultures: A549 cells were grown in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mML-glutamine, 100 units/mL penicillin G and 100 μg/mL streptomycin to 80%confluence. Cells were dispersed with Trypsin-EDTA and seeded onto glasscoverslips or 8 well confocal imaging chambers and grown to confluencein the presence of DMEM.

Confocal Analysis: Bacteria were prepared and counterstained with Syto82 nucleic acid stain (Invitrogen, Calif., USA) in a shaking heat blockat 37° C. and 350 rpm for 20 minutes. They were co-incubated with theprobe at required concentration in a sealed POC mini chamber, or 8 wellconfocal chamber. When required the glass coverslip for the POC chamberwas coated in fibronectin (for neutrophil experiments) or poly-d-lysine(for bacteria and cell lines) and incubated with cells with one hour at37° C. to allow adherence prior to bacterial innoculation. Analysis waswith ImageJ. Briefly, the Syto channel was automatically thresholded(Huang) and an ROI generated from this. The mean fluorescence intensityon the probe channel within this ROI was quantified. Data presentedrepresents the mean of three separate fields of view.

Flow Cytometry: Bacteria were prepared and counterstained with Syto 82nucleic acid stain (Invitrogen, Calif., USA) in a shaking heat block at37° C. and 350 rpm for 20 minutes. Bacteria were washed in PBS×3, andprobe (50 μl) added in 50 ul OD₅₉₅ 1 od bacteria. This was diluted to500 uL and analysed using BD FACS Calibur using FL-1 and FL-2 channels,with 10,000 events. Analysis was with FlowJo software following gatingon the FL-2 channel.

Lung Harvesting and pCLE procedure: From a cohort of surplus stock eweswhich were destined for cull, one ewe was identified and terminallyeuthanized with an overdose of anaesthetic. Death was confirmed and thetrachea was identified and clamped in situ. The thoracic cavity was thenaccessed and the lungs were freed from surrounding tissues and organsand the heart/lung was removed en block. The right pulmonary artery wasidentified, cannulated and perfused with 1000 ml 0.9% NaCl. Once fillingof the left ventricle was confirmed an incision was made to allow freedrainage and perfusion continued until the drainage from the left atriumwas clear. The trachea was intubated with an 8.0 endotracheal tubeimmediately following clamp release. The lungs were placed in a neonatalincubator with an ambient temperature of 37° C. and humidity of 65% andventilated using a Pressure Controlled Ventilator (Breas Vivo PV 403).Ventilator setting was adjusted to aid maximal parenchymal recruitmentand aiming to achieve tidal volume >1 litre. Following 1 hour of optimalventilation, bronchoscopy was undertaken and individual segments wereidentified and instilled with 2 ml of bacteria or PBS control. Followinginstillation a separate sheath (ERBE) was introduced and the probe wasinstilled. Then the probe-based Confocal Laser Endomicroscopy (pCLE)fibre was passed down the working channel and the segment was imaged.For BALF, the bronchoscope was wedged and 20 ml of 0.9% NaCl instilledand carefully withdrawn with lavage yields of 40-50%. Control segmentswere anatomically distinct and/or in the contralateral lung. Thebronchoscope was decontaminated between each segment imaged.

Haemolysis Assay: Erythrocytes were isolated from freshly drawn,anticoagulated human blood and resuspended to 20 vol % in PBS (pH 7.4).In a 96-well microtiter plate, 100 μl of erythrocyte suspension wasadded to 100 μl of NLLP solution in PBS (prepared by 1:2 serialdilutions) or 100 μl of PBS in the case of negative controls.One-hundred percent haemolysis wells contained 100 μl of red cellsuspension with 100 μl of 0.2 vol % Triton X-100. The plate wasincubated for 1 h at 37° C., and then each well was diluted with 150 μlof PBS. The plate was then centrifuged at 1,200 g for 15 min, 100 μl ofthe supernatant from each well was transferred to a fresh microtiterplate, and A350 was measured. Percentage of haemolysis was determined as(A−A0)/(Atotal−A0)×100, where A is the absorbance of the test well, A0the absorbance of the negative controls, and Atotal the absorbance of100% haemolysis wells, all at 350 nm on a Biotek plate reader.

MALDI-TOF: Probe was added to saline or pooled BALF from patients withALI incubated for 30 minutes. A ZipTip (C-18, 0.2 μL) with 5 μL MeCN(with 0.1% TFA as an additive) followed by 20 μL of H2O was washed. TheZipTip was loaded with the sample, washed and eluted into 5 μL of 80%aq. MeCN (with 0.1% TFA as an additive). The sample was analysed byMALDI-TOF (PerSeptive Biosystems Voyager DE™STR MALDI-TOF massspectrometer (Applied Biosystems, Foster City, Calif.)).

Statistical Analysis: All experiments were performed at least threetimes unless otherwise stated and results expressed as mean±SEM. Datawas analysed by unpaired t-test or ANOVA, significance was determined asp<0.05 (GraphPad Prism).

REFERENCES

1. M. Ternon, J. J. Diaz-Mochon, A. Belsom, M. Bradley, Tetrahedron,2004, 60, 8721

2. H. J. Knölker, T. Braxmeier, G. Schlechtingen, Angew. Chem. Int. Ed.,1995, 34, 2497

3. E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook,Analytical Biochemistry, 1970, 34, 595-598.

LISTING OF RELEVANT SEQUENCES

Ubiquicidin (full) SEQ ID NO. 1KVHGSLARAG KVRGQTPKVA KQEKKKKKTG RAKRRMQYNR RFVNVVPTFG KKKGPNANSUbiquicidin (UBl₂₉₋₄₁) SEQ ID NO. 2 TGRAKRRMQY NRRUbiquicidin (UBl_(Nle)) SEQ ID NO. 3 TGRAKRRNleQY NRR PolymyxinSEQ ID NO. 4

Dab = L-α-γ-Diaminobutyric acid Polymyxin B1 SEQ ID NO. 5

Polymyxin B2 SEQ ID NO. 6

1. A probe comprising a core and a plurality of probe elements; eachprobe element within the plurality of probe elements extending from thecore and comprising a fluorophore and a binding moiety, wherein thebinding moiety is a bacteria binding moiety and selectively binds to atleast some bacteria, and does not bind to animal cells, such asmammalian cells, or does so weakly, or wherein the binding moiety is afungi binding moiety, or wherein the binding moiety may selectively bindto at least some bacteria and to at least some fungi, and not bind toanimal cells, such as mammalian cells, or does so weakly.
 2. The probeaccording to claim 1, wherein the plurality of probe elements compriseat least three probe elements.
 3. (canceled)
 4. (canceled)
 5. (canceled)6. The probe according to claim 1, wherein, the binding moiety of one ormore of the probe elements is a ubiquicidin moiety, such as the fulllength ubiquicidin (SEQ ID NO.1) or a fragment or variant thereof. 7.The probe according to claim 6, wherein the binding moiety is theubiquicidin fragment of amino acids 29 to 41 (UBI₂₉₋₄₁, SEQ ID NO.2). 8.The probe according to claim 6, wherein the binding moiety is UBI₂₉₋₄₁comprising a substitution of a norleucine amino acid for the originalmethionine amino acid (UBI^(29-41NIe), SEQ ID NO.3).
 9. (canceled) 10.(canceled)
 11. The probe according to claim 1, wherein each probeelement within the plurality of probe elements comprises the samefluorophore, or wherein each probe element comprises differentfluorophores.
 12. (canceled)
 13. The probe according to claim 1, whereinthe fluorophore of each probe element within the plurality of probeelements is an environmentally sensitive fluorophore, such that theintensity or quantum yield of fluorescence of the fluorophore depends onthe surroundings of the fluorophore.
 14. The probe according to claim13, wherein the quantum yield or intensity of the fluorophore is higherin a hydrophobic environment, such as within a cell membrane, whereinthe fluorophore of one or more of the probe elements within theplurality of probe elements is 7-nitrobenz-2-oxa-1,3-diazole (NBD),malachite green, a styryl-based dye, Cascade Yellow, prodan (aka1-Propanone, 1-(6-(dimethylamino)-2-naphthalenyl), Dansyl (aka.5-(dimethylamino)naphthalene-1-sulfonyl), Dapoxyl, PyMPO (aka.1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-Methoxyphenyl)Oxazol-2-yl)Pyridinium),pyrene and diethylaminocumarin, or derivatives or variants thereof. 15.(canceled)
 16. (canceled)
 17. The probe according to claim 14, whereinthe fluorophore of each probe element within the plurality of probeelements is a NBD moiety.
 18. The probe according to claim 1, whereinthe fluorophore of one or more of the probe elements within theplurality of probe elements has a long fluorescent lifetime and the longfluorescent lifetime of the fluorophore allows the detection of theprobe over background autofluorescence.
 19. The probe according to claim18, wherein the fluorophore is azadioxatriangulene (ADOTA) dye ordiazaoxatriangulene (DAOTA), or derivatives thereof.
 20. (canceled) 21.The method according to claim 1, wherein the fluorophore is detectablevia photoacoustic imaging.
 22. The probe according to claim 1, whereinone or more fluorophores of adjacent probe elements within the pluralityof probe elements may quench the fluorescence of one or more of theother fluorophores of the probe, wherein the self-quenching is preventedor reduced when the environment of the probe is changed.
 23. (canceled)24. The probe according to claim 1, wherein the binding moiety is at thedistal end of the probe element furthest from the core.
 25. (canceled)26. (canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. Theprobe according to claim 1, wherein the probe comprises a quencherconnected to the core by a cleavable linker; the fluorophore of one ormore of the probe elements within the plurality of probe elements issubstantially fluorescently quenched by the quencher when the core isconnected to the quencher by the cleavable linker; wherein the core, andthereby the plurality of probe elements, is separated from the quencherwhen the cleavable linker is cleaved.
 31. (canceled)
 32. The probeaccording to claim 1, wherein the probe comprises a quencher connectedto the core, and the plurality of probe elements are connected to thecore by a cleavable linker; the fluorophore of one or more of the probeelements within the plurality of probe elements is substantiallyfluorescently quenched by the quencher when the or each probe elementwithin the plurality of probe elements are connected to the core, andthereby the quencher, by the cleavable linkers; wherein the core, andthereby the quencher, is separated from the plurality of probe elementswhen the cleavable linkers are cleaved.
 33. The probe according to claim1, wherein the cleavable linker comprises an enzyme cleavable peptidesequence, and the linker is cleaved when a cleaving enzyme cleaves theenzyme cleavable peptide sequence.
 34. (canceled)
 35. (canceled) 36.(canceled)
 37. (canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled)41. (canceled)
 42. (canceled)
 43. (canceled)
 44. (canceled) 45.(canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. The probeaccording to claim 1, wherein the probe has one of the followingstructures:

where L=spacer group (e.g. C₃-C₁₀ alkyl, ((CH₂)₂O)_(x) where x=1-6)F=fluorophore (e.g. NBD, fluorescein) B=binding moiety (e.g. ubiquicidinmoiety, polymyxin moiety) C=core (e.g. carbon, —C(CH₂O(CH₂)₃)₃,N₂CONHC(CH₂O(CH₂)₃)₃)
 50. The probe according to claim 49, wherein theprobe comprises three probe elements connected to a core each probeelement comprising NBD-(CH₂)₅CO-UBI_(NIe).
 51. The probe according toclaim 49, wherein the probe comprises three probe elements connected toa core each of the three probe elements comprisingNBD-(CH₂)₂O(CH₂)₂OCH₂-PMX.
 52. The probe according to claim 49, whereinthe probe has the structure:


53. A method of detecting bacteria and/or fungi in a target area, themethod comprising the steps: (1) providing a first probe according toclaim 1; (2) delivering the first probe to the target area; (3)illuminating the target area with an appropriate wavelength of light toexcite the fluorophores of the first probe; and (4) determining whetherthe first probe has labelled bacteria and/or fungi within the targetarea.
 54. (canceled)
 55. (canceled)
 56. (canceled)
 57. (canceled) 58.(canceled)
 59. (canceled)
 60. The method according to claim 53, whereinthe method comprises the steps of: providing a second probe according toclaim 1 comprising a binding moiety that specifically binds to asubpopulation of bacteria; delivering the second probe to the targetarea; and illuminating the target area with an appropriate wavelength oflight to excite the or each fluorophore of the second probe.
 61. Themethod according to claim 60, wherein the first probe comprises aubiquicidin moiety, such as full length ubiquicidin, or a fragment orvariant thereof as the binding moiety.
 62. The method according to claim60, wherein the second probe comprises a polymyxin moiety, such as fulllength polymyxin or a fragment or variant thereof, as the bindingmoiety, and the second probe may selectively bind to gram negativebacteria.
 63. The method according to claim 62, wherein the first probecomprises a ubiquicidin moiety and the method allows the identificationof sterile inflammation, bacterial and/or fungal inflammation, and gramnegative bacterial infection (and by inference, gram positive bacterialinfection).
 64. (canceled)
 65. (canceled)
 66. (canceled)
 67. A kit ofparts comprising a first probe according to claim 1 comprising at leastone binding moiety that specifically binds to bacteria and/or fungi, anda second probe according to claim comprising at least one binding moietythat specifically binds to gram-negative or gram-positive bacteria and asuitable buffer within which the probe may be dispersed.
 68. (canceled)69. The kit of parts according to claim 67, wherein the first probecomprises at least one ubiquicidin moiety, and the second probecomprises at least one polymyxin moiety.